Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland.

Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable. In all experimental groups, administration of IPR led to a rapid, transient increase in the c-fos mRNA level. Propranolol blocked the IPR effect, while treatment with IPR and cycloheximide led to superinduction. We observed no correlation between the effect of IPR on cell replication or organ growth and stimulation of c-fos expression, and conclude that the latter is the result of beta-adrenergic receptor-IPR interaction. The c-fos protein was localized immunocytochemically in both the cytoplasm and the nuclei of acinar cells and in the nuclei of duct cells.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.

Density-dependent nerve growth factor regulation of Gs-alpha RNA in pheochromocytoma 12 cells.

Nerve growth factor (NGF) affects levels of the alpha subunit of the stimulatory G protein (Gs-alpha) in pheochromocytoma 12 cells in a bidirectional, density-dependent manner. Cells grown at high density responded to NGF treatment with increased levels of Gs-alpha mRNA and protein. Conversely, in cells grown in low-density cultures, levels of this mRNA were lowered by NGF treatment.

c-fos mRNA levels are increased by the cellular stressors, heat shock and sodium arsenite.

Rapid, transient expression of the c-fos proto-oncogene is induced by the beta-adrenergic agonist, isoproterenol, in vivo and by numerous factors promoting growth and differentiation in cultured cells. We wanted to determine whether cellular stressors, which are known to induce expression of the gene encoding the major heat shock protein, hsp 70, also induced expression of the c-fos gene. Our findings demonstrate that c-fos mRNA levels increase transiently under conditions of heat stress or sodium arsenite treatment which induce expression of hsp70 mRNA in cultured cell lines. When 3T3 cells are heat shocked in the presence of amiloride, an inhibitor of Na+/H+ exchange, the induction of c-fos mRNA is partially inhibited, whereas that of hsp70 is somewhat enhanced. However, neither response requires ongoing protein synthesis. In fact, dramatic superinduction of c-fos mRNA is observed in cells which are heat shocked in the presence of the protein synthesis inhibitor, anisomycin. A comparison of relative rates of protein synthesis and c-fos mRNA levels during either heat shock or sodium arsenite treatment suggests that the transient suppression of protein synthesis accompanying these treatments may be one factor responsible for the observed c-fos mRNA induction.

Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro.

Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus. This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+. It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride. Alterations in membrane permeability and in ion fluxes and/or perturbation in membrane phospholipids may play a role in this transitional activation of the c-fos gene expression in incubated tissue slices in which the cells are not viable and undergo a necrobiotic process.

Epidermal growth factor gene expression is regulated differently in mouse kidney and submandibular gland.

The concentration of the peptide mitogen epidermal growth factor (EGF) is hormonally and developmentally regulated in the granular convoluted tubule cells of the mouse submandibular gland. Using a labeled EGF nucleic acid probe, we have demonstrated that submandibular gland EGF mRNA concentrations increase during postnatal development of the gland and after the administration of testosterone or thyroid hormone. Recently, it was reported that EGF mRNA is present in kidney as well as a number of other mouse tissues. A comparison of EGF gene regulation in submandibular gland and kidney revealed that kidney EGF mRNA levels also increase during the postnatal period. Opposite sex differences were observed, with submandibular gland levels being about 16-fold higher in the male than in the female and kidney levels being 2- to 4-fold higher in the female than in the male. Renal EGF mRNA concentrations are less responsive to hormones than those in the submandibular gland. Renal EGF was localized immunocytochemically to the cells of distal convoluted tubules.

Effect of advanced age on the induction by androgen or thyroid hormone of epidermal growth factor and epidermal growth factor mRNA in the submandibular glands of C57BL/6 male mice.

We have compared the responsiveness of the submandibular glands of mature (12 month old) and senescent (26-28 month old) male C57BL/6 mice to dihydrotestosterone (DHT) or triiodothyronine (T3) in terms of steady state levels of epidermal growth factor (EGF) protein and EGF mRNA. Northern blot analyses did not disclose any differences with age in the apparent sizes of EGF mRNA species. In untreated animals, submandibular glands of 26-28-month-old mice contained approximately 50% less EGF, and 75% less EGF mRNA than those of 12-month-old males. With advanced age, there was a 20% reduction in the absolute volume of the granular convoluted tubule (GCT) compartment, which is the exclusive site of EGF and EGF mRNA in the gland. In general, GCTs of old mice were composed of smaller cells with fewer secretion granules, but there was considerable cell-to-cell variation. In addition, there was greater variation in the intensity of immunocytochemical staining for EGF in senescent GCT cells, which also gave a lower and more variable in situ hybridization signal for EGF mRNA. After hormonal stimulation for 1 week with either tri-iodothyronine (T3) or dihydrotestosterone (DHT), EGF protein concentration in the glands was induced to the same level at both ages. However, EGF mRNA was 50% less abundant in old hormonally stimulated glands, compared to similarly treated young ones. Although many GCT cells in treated glands of senescent males respond to hormonal stimulation by increases in size and in content of secretion granules, there was cell-to-cell variation in responsiveness, especially after treatment with T3. These findings indicate that the decreases seen in the entire gland in EGF and EGF mRNA are caused by a wide-spread deterioration of the GCT cells themselves, which apparently can be reversed in many but not all GCT cells by stimulation with supraphysiologic doses of either T3 or DHT.

Expression of immediate early genes after treatment of human astrocytoma cells with radiation and taxol.

PURPOSE: The promising chemotherapeutic agent, taxol, has been shown to sensitize the G18 line of human astrocytoma cells to ionizing radiation. The present studies were performed to identify specific changes in gene expression associated with this altered sensitivity. METHODS AND MATERIALS: The radioresistant, grade 3 human astrocytoma cell line, G18, was exposed for varying periods of time to treatment with taxol, tetradecanoyl phorbol acetate (TPA), serum, isoproterenol, dibutyryl cyclic adenosine monophosphate, or ionizing radiation alone or in combination with taxol pretreatment. Ribonucleic acid samples from the cells were monitored for the expression of a group of immediate early genes (IEGs), including c-fos, c-jun, TIS1, TIS7, TIS8, TIS11 and TIS21, by northern blot hybridization analysis. RESULTS: Transient immediate early gene induction was observed after treatment of G18 cells with tetradecanoyl phorbol acetate, serum, isoproterenol, or ionizing radiation, but not after treatment with taxol. Of the seven immediate early genes analyzed, all but TIS7 were found to be inducible by one or more of the treatments. Only TIS8 (also known as egr-1 or zif268) was significantly inducible by radiation, and this transient induction was decreased by at least four-fold by pretreatment for 24 hr with a dose of taxol that was previously shown to block 96.5% of the cells in G2/M and enhance radiosensitivity. CONCLUSION: The products of immediate early genes, which are induced transiently in cells in response to a variety of treatments, including growth factors, neurotransmitters, and irradiation with UV light or X rays, are thought to initiate a cascade of genetic responses to alterations in cellular environment. The present results demonstrate a dramatic attenuation in one immediate early gene response in association with a treatment that enhances radiosensitivity in a refractory human brain tumor line.

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

Adrenergic receptors mediate changes in c-fos mRNA levels in brain.

Recent evidence supports a role for transient c-fos expression as one step in signalling pathways by which membrane receptor-ligand interactions are transduced into appropriate intracellular responses. The activity of adrenergic receptors is mediated by second messenger systems which include ion fluxes, changes in cAMP concentration and enhanced phosphoinositide turnover. In order to determine if C-fos induction was also a step in adrenergic signal transduction in the brain, we performed in vivo studies with drugs specific for different adrenergic receptor types. Unexpectedly, we found that the stress associated with a single intraperitoneal (i.p.) injection of drug vehicle produced a transient increase (averaging 4.0-fold) in c-fos mRNA levels in rat brain. Injection of the alpha 2-adrenoreceptor antagonist, yohimbine, produced a transient increase which was larger in magnitude (averaging 9.6-fold) and longer in duration than that produced by injection of the drug vehicle alone. In experiments designed to ask whether either of these inductions was mediated by specific types of adrenergic receptors, we found that the alpha 2- and beta-adrenoreceptors were involved in both responses, while the alpha 1-receptor played a role in mediating the yohimbine induction, but no detectable role in the solvent induction. One hypothesis consistent with our results is that the norepinephrine (NE) released due to the stress associated with an i.p. injection interacts with postsynaptic beta-adrenergic receptors, resulting in the observed c-fos mRNA induction. When the negative feedback effect of NE, mediated by presynaptic alpha 2-receptors, is blocked by yohimbine, the postsynaptic response is enhanced and prolonged.

Activation of adenosine receptors induces c-fos, but not c-jun, expression in neuron-glia hybrids and fibroblasts.

Extracellular adenosine acts through specific cell surface receptors to modulate numerous physiological processes in both the CNS and peripheral tissues (e.g. neurotransmitter release and blood flow). Activation of A1 or A2 adenosine receptors leads to decreased or increased intracellular cAMP levels, respectively. Fos and Jun are nuclear proto-oncogene products, which, like cAMP, appear to act as intermediates in a number of signal transduction pathways. Since increases in both adenosine release and Fos and Jun expression occur in the brain following seizures, we wanted to determine whether Fos and Jun induction might occur as a result of adenosine receptor activation. 3T3 fibroblasts and NG108-15 neuroblastoma-glioma hybrid cells were chosen for study, since they were known to respond to adenosine agonists with changes in cAMP levels. The membranes of NG108-15 cells were shown to have A2-like binding activity in a competitive binding assay. Cultures of each cell line were treated with the adenosine agonists, CHA (A1-selective) and NECA (non-selective adenosine agonist). Both lines responded with a concentration-dependent transient increase in c-fos, but not c-jun, mRNA content after treatment with either agonist. The kinetics of the response were much more rapid for 3T3 cells (peak between 15 and 30 min) than for NG cells (peak between 60 and 90 min). The slower, more prolonged response in the NG108-15 cells is more similar to the time interval between adenosine release and the peak of c-fos mRNA induction in brains of animals following the administration of seizure-promoting drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate early gene induction after neonatal hypoxia-ischemia.

Immediate early gene (IEG) products, such as FOS and JUN, may partially mediate the long-term transcriptional response of CNS cells to specific changes in their environment. To determine whether IEG products might be involved in the immature brain's response to hypoxia-ischemia (H-I), 7-day-old rat pups were subjected to unilateral common carotid artery ligation followed by 3 h of hypoxia (8% O2/92% N2) at 37 degrees C, which results in pathological changes only in specific regions of the hemisphere ipsilateral to ligation. Time course experiments were performed, in which animals were sacrificed between 1 and 24 h after H-I. RNAs from several brain regions were analyzed by Northern blot hybridization for their relative concentrations of nine IEG mRNAs (c-fos, c-jun, junB, TIS 1 (nur77), TIS7, TIS8 (zif268), TIS10, TIS11, and TIS21). Induction of all IEGs, except TIS7 and TIS10, was observed in ipsilateral forebrain, and, less frequently, in contralateral forebrain, at 1, 2, and 3 h post-hypoxia. In some animals, lower levels of expression were also detected at 4, 18 and 24 h. With minor exceptions, co-induction of all seven IEGs was observed in a given RNA sample. Induction of two other mRNAs, representing the heat shock and astrocytic responses, were also observed. Hsp70 mRNA levels were increased only in the brains of animals exhibiting IEG induction. However, hsp70 induction was confined to the ipsilateral forebrain, implying a more direct relationship between its expression and permanent morphological damage. GFAP mRNA induction occurred predominantly in ipsilateral forebrain samples at 18 and 24 h post-hypoxia. Levels of B-actin and ubiquitin mRNAs were relatively constant in the same RNA samples. In control experiments c-fos mRNA induction was not detected after sham ligation with hypoxia, ligation with sham hypoxia, or hypoxia alone. These results suggest that the immature brain is highly responsive to H-I at the level of gene expression, involving at least three different rapid response systems.

Variations in c-fos gene expression during rat brain development.

Expression of the c-fos proto-oncogene has been associated with mitosis or differentiation in a number of tissue culture model systems. We have studied the expression of this gene during in vivo brain development in the rat. Our results demonstrate that very low levels of c-fos mRNA are detectable during the period of development characterized by rapid mitosis, whereas much higher concentrations of c-fos mRNA are found in the brains of older neonatal animals and adults. Therefore, although c-fos could be participating in the regulation of mitosis during early postnatal development of the brain, it is also likely to play an important role in mature brain tissue.

Localization of c-fos, c-jun, and hsp70 mRNA expression in brain after neonatal hypoxia-ischemia.

The sites of expression of early response mRNAs were determined in the brains of 7-day-old rat pups exposed to unilateral carotid artery ligation followed by 3 h of hypoxia. Pups were sacrificed after recovery periods ranging from 10 min to 24 h. In agreement with our previous northern blot analysis, in situ hybridization of coronal brain sections to probes for c-fos, c-jun, and heat-inducible hsp70 revealed a marked induction and subsequent disappearance of all three mRNAs during this time period. We observed co-localization of the 2 immediate early gene (IEG) mRNAs, c-fos and c-jun, which encode proteins that act in combination to regulate subsequent gene expression. These mRNAs were expressed in all regions known to be vulnerable to permanent injury in this model, such as the cortex, hippocampus, and striatum, as well as in other regions that are spared from permanent damage, such as contralateral cortex and lateral ventricular neuroepithelium. The temporal and regional co-localization of c-fos and c-jun suggests that the transcriptional regulatory activity of their protein products could play a role in plasticity associated with death or recovery from injury in the immature brain. Hsp70 mRNA expression was induced in nearly all of the animals that were positive for IEG mRNAs. Although the most frequent site of expression for all three mRNAs was the ipsilateral cerebral cortex, hsp70 expression was restricted to the ipsilateral hemisphere and absent from a number of structures that were positive for c-fos and c-jun. In addition, the patterns of expression of hsp70 within specific structures frequently differed from those of the IEGs, implying that although both cellular early response systems are activated in this model, their specific functions are carried out within different microenvironments.

Beta-adrenergic treatment of C6 glioma cells produces opposite changes in c-fos and c-jun mRNA levels.

The AP1 transcriptional complex is a heterodimer composed of proteins encoded by the fos and jun proto-oncogene families. Changes in the concentration and composition of AP1 occur after cells are perturbed in a variety of different ways (Curran, in Reddy et al., eds. "The Oncogene Handbook," Amsterdam: Elsevier, pp 307-325, 1988; Sonnenberg et al., Neuron 3:359-365, 1989). Transient changes in AP1 content presumably result in altered expression of AP1-regulated target genes, that help to mediate the cell's long-term response to changes in its environment. One factor that may be important in determining which target genes are regulated by AP1 in a given context is the identity of the jun family member present in the complex (Chiu et al., Cell 59:979-986, 1989; Schutte et al., Cell 59:987-997, 1989). Fos induction has been demonstrated after binding of beta-adrenergic ligands to their cell surface receptors (Barka et al., Mol Cell Biol 6:2984-2989, 1986; Gubits et al., Mol Brain Res 6: 39-45, 1989; Arenander et al., J Neurosci Res 24: 107-114, 1989; Mocchetti et al., Proc Natl Acad Sci USA 86:3891-3895, 1989). However, the response of the jun gene family to this treatment has not been reported. We have therefore examined the effect of beta-adrenergic receptor activation on the expression of c-fos, c-jun, and junB mRNA levels in C6 glioma cells. Our results indicate that c-fos and junB mRNA levels are increased by 52- and 2.7-fold, respectively, after 45 min of isoproterenol (IPR) treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental and hormonal regulation of alpha 2u-globulin gene transcription.

Hepatic alpha 2u-globulin protein and RNA levels are under developmental and complex multihormonal control. The present studies directly evaluate the degree to which this regulation is transcriptional. alpha 2u-Globulin transcription was determined by measuring nuclear runoff RNA in vitro, and tissue alpha 2u-globulin mRNA levels were measured by dot blot hybridization. These studies reveal that (i) in male rats the transcriptional rate of the alpha 2u-globulin genes increases during postnatal development; (ii) no alpha 2u-globulin transcription is detectable in hepatic nuclei derived from hypophysectomized rats; (iii) growth hormone and glucocorticoid are both absolutely required, and glucocorticoid can replace androgen for alpha 2u-globulin gene transcription in the livers of hypophysectomized male rats; and (iv) chronic treatment of mature male rats with estrogen results in a progressive decrease in the hepatic transcription of alpha 2u-globulin genes. In all instances changes in the transcriptional rate of alpha 2u-globulin genes paralleled the tissue level of alpha 2u-globulin RNA. Thus transcriptional control predominates in regulating hepatic alpha 2u-globulin RNA levels.

Apoptosis in substantia nigra following developmental striatal excitotoxic injury.

We have previously observed that an axon-sparing injury to the developing striatum induced by the excitotoxin quinolinate results in a decrease in dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the adult. This decrease occurs in the absence of direct injury to the SNpc. As the striatum is a major target for the SNpc dopaminergic system, we have hypothesized that a decrease in the size of the striatal target during development may result in an induced regressive event in the SNpc, similar to what has been described for many developing neural systems with peripheral targets. We have examined by morphologic and biochemical means the time course and character of cell death in SN following a unilateral striatal lesion with quinolinate in immature rats. The striatal lesion is associated with an induced cell death event in the ipsilateral SN, observed first in SNpc and then in SN pars reticulata. The morphologic characteristics of the dying cells were typical of apoptosis. Immunostaining for tyrosine hydroxylase indicated that some of the apoptotic cells in the SNpc were dopaminergic. We conclude that developmental striatal excitotoxic injury is associated with induced apoptotic cell death in SN.

Altered genetic response to beta-adrenergic receptor activation in late passage C6 glioma cells.

Previous studies have demonstrated variability in the phenotype of rat C6 glioma cells. In the present study, we compared morphology, growth rate, and beta-adrenergic regulation of gene expression in early (P39-47) and late (P55-90) passage C6 cells. Morphological changes were observed in five independently derived, late passage populations. In four of the five, the untreated cells were more polygonal than the fibroblast-like parental cells, and only a small fraction exhibited process outgrowth after dbcAMP treatment. Untreated cells from the fifth late passage population had longer cytoplasmic processes than parental cells and responded to dbcAMP with further process outgrowth. All late passage populations had shorter generation times than the parental cells. In early passage cells, treatment with the beta-adrenergic agonist, isoproterenol (IPR), resulted in an increase in c-fos mRNA and a decrease in c-jun mRNA (Gu-bits RM, Yu H: J Neurosci Res, 30:625-630, 1991). Both of these immediate early gene responses were irreversibly lost between P50 and P55. Additional differences in basal or IPR-induced mRNA levels were observed for beta-APP, GFAP, NGF, and PPE, but not for a number of other mRNAs. These results are discussed in relationship to previously described differences in the ability of early and late passage C6 cells to accumulate cAMP (Mallorga P, et al.: Biochim Biophys Acta 678:221-229, 1981).

Characterization of sarcomeric myosin heavy chain genes.

Myosin heavy chain is encoded by a large multigene family. Using pMHC-25, a recombinant cDNA clone isolated from the rat myogenic cell line L6E9, four members of this family in the rat have been isolated and shown to be tissue-specific and developmentally regulated. The coding regions of these genes share regions of homology interspaced with regions of non-homology. Detailed analysis of one embryonic and one adult myosin heavy chain gene shows that the coding sequences are interrupted by numerous intervening sequences whose number, size, and distribution do not appear to be conserved in the same organism or between species.

Sarcomeric myosin heavy chain is coded by a highly conserved multigene family.

pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum of seven genes, some of which are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.