Long term effect of RU24722 on tyrosine hydroxylase in the rat locus coeruleus: differential effects of two enantiomeric forms

In the present paper we analyzed c-fos and zif/268 expression in rat primary astroglial cell cultures after treatment with Platelet-activating Factor (PAF) and its 2-O-methyl-analogue, 1-O-octadecyl-2-O-methoxy-glycero-3-phosphocholine (ET-18-OCH₃). Both compounds, at a dose (2 μM) that did not produce toxic effects on astroglial cells, induced a rapid and transient increase of c-fos and zif/268 mRNA level. Pretreatment of astroglial cells with the PAF antagonist BN50730 (5 μM) 10 min prior to the addition of alkyl-phospholipids almost completely prevented the activation of the immediate early genes. On the contrary triazolam, another PAF inhibitor, did not block PAF induced gene expression when added to the medium at 5 μM concentration. ET-18-OCH₃ effect on gene expression is blocked by the same antagonist (BN50730) which is effective in inhibiting PAF effect on astrocytes, suggesting that both substances act through the same binding site.

Results obtained support the view that astroglial cells are a cellular target for this lipid mediator, and, like macrophages, respond to its methoxy-analogue.     

Platelet-activating factor and its methoxy-analogue et-18-OCH3 stimulate immediate early gene expression in rat astroglial cultures

In the present paper we analyzed c-fos and zif/268 expression in rat primary astroglial cell cultures after treatment with Platelet-activating Factor (PAF) and its 2-O-methyl-analogue, 1-O-octadecyl-2-O-methoxy-glycero-3-phosphocholine (ET-18-OCH₃). Both compounds, at a dose (2 μM) that did not produce toxic effects on astroglial cells, induced a rapid and transient increase of c-fos and zif/268 mRNA level. Pretreatment of astroglial cells with the PAF antagonist BN50730 (5 μM) 10 min prior to the addition of alkyl-phospholipids almost completely prevented the activation of the immediate early genes. On the contrary triazolam, another PAF inhibitor, did not block PAF induced gene expression when added to the medium at 5 μM concentration. ET-18-OCH₃ effect on gene expression is blocked by the same antagonist (BN50730) which is effective in inhibiting PAF effect on astrocytes, suggesting that both substances act through the same binding site.Results obtained support the view that astroglial cells are a cellular target for this lipid mediator, and, like macrophages, respond to its methoxy-analogue.     

Acute intrarenal infusion of ANG II does not stimulate immediate early gene expression in the kidney

ANG II is capable of stimulating expression of immediate early genes such as egr-1 and c-fos in a variety of cultured cells, including cells of renal origin. To investigate whether ANG II can stimulate early growth response gene expression in vivo, we studied the effects of acute renal artery infusion of low-dose ANG II (2.5 ng small middle dot kg(-1) small middle dot min(-1)) or vehicle on the renal expression of c-fos and egr-1 genes in rats. ANG II infusion for 30 or 240 min decreased renal vascular conductance by approximately 13 and 8%, respectively, compared with the vehicle group. Expression of the early growth response genes c-fos and egr-1 was analyzed using Northern blot hybridization. No significant upregulation of c-fos or egr-1 mRNA levels was detected in rats that received ANG II for either 30 or 240 min, compared with the vehicle groups. We conclude that ANG II, at doses that cause significant physiological effects, does not increase the renal expression of c-fos or egr-1 genes over periods of up to 4 h in vivo.     

Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells.

Platelet-activating factor (PAF) has recently been demonstrated to be metabolized by B lymphocytes and to cause enhancement of Ig synthesis by Ig-secreting B lymphoblastoid cell lines. We have now examined some of the early activation events triggered by PAF binding to three Ig-secreting B cell lines, LA350 (IgM secreting), HSCE- (IgG secreting), and U266 (IgE secreting). After addition of 10(-7) to 10(-11) M PAF, but not equimolar concentrations of the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The increases in [Ca2+]i resulted from both the release of Ca2+ from internal stores as well as transmembrane Ca2+ uptake. Addition of PAF triggered the rapid hydrolysis of phosphatidylinositol bisphosphate and accumulation of inositol phosphates. PAF also increased expression of the cell cycle-active genes c-fos and EGR2 in a dose-dependent fashion. The stimulated increases in [Ca2+]i and phosphatidylinositol bisphosphate hydrolysis and the increases in gene expression were all inhibited by the specific PAF receptor antagonist Web 2086. The LA350 cell line (which expresses surface IgM) was also shown to increase [Ca2+]i after addition of anti-IgM antibodies. Sequential addition of PAF or anti-IgM antibody in either order failed to reveal any evidence for heterologous desensitization. Furthermore, the PAF receptor antagonist did not affect anti-IgM induced changes in [Ca2+]i. These data provide evidence for the presence of functional PAF receptors on B lymphoblastoid cells and indicate a potential role for PAF in the regulation of B cell activation.     

Induction of immediate early genes by Ca2+ influx requires cAMP-dependent protein kinase in PC12 cells

Agents that activate cAMP-dependent protein kinase (PKA) as well as agents that increase intracellular calcium induce the expression of certain immediate early genes (IEGs). Recently, it has been demonstrated that the same cis-acting element in the 5' region of the c-fos gene has the ability to mediate both cAMP- and calcium-induced c-fos expression in PC12 cells (Sheng, M., McFadden, G., and Greenberg, M. (1990) Neuron 4, 571-582). Here we demonstrate that both cAMP- and calcium-mediated induction of c-fos and egr1 are dependent on PKA activity. Addition of either depolarizing concentrations of KCl or the calcium ionophore, ionomycin, to PC12 cells increased the expression of both c-fos and egr1, but these inductions were dramatically reduced in three PKA-deficient cell lines, 123.7, AB.11, and A126-1B2. Furthermore, pretreatment of PC12 cells with 20 microM H89, a specific inhibitor of PKA, inhibited forskolin, dibutyryl cAMP, and KCl-induced c-fos and egr1 induction, while having no effect on NGF induction. Likewise, in the PKA-deficient cells, NGF or an activator of protein kinase C induced c-fos and egr1 normally. To determine if PKA deficiency modifies the ability of Ca2+ to activate calcium-dependent kinases, autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in response to Ca2+ influx was determined. In parental PC12 cells, PC12 cells pretreated with H89, and PKA-deficient cell lines, CaM kinase was activated equivalently in response to KCl depolarization. These results suggest that PKA is not required for Ca(2+)-induced increase in CaM kinase activity and that the induction of IEGs in response to Ca2+ influx is PKA-dependent. Thus, the requirement for PKA resides at a point distal to the activation of calmodulin-dependent processes.     

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF- I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early- response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H- ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.     

Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells

During pregnancy, stretch of the uterus, imposed by the growing fetus, is an important signal for the induction of genes involved in the onset of labor. In this study, the expression of activator protein-1 (AP-1) family mRNAs in response to in vitro stretch was investigated in myometrial cells. Rat primary myometrial smooth muscle cells were plated onto collagen I-coated Flex I culture plates and subjected to 25% static stretch on day 4 of culture. Static stretch induced an increase in the expression of c-fos, fosB, fra-1, c-jun, and junB. The expression of both c-fos and junB was maximally induced at 30 min by static stretch. The peak induction for fosB and c-jun occurred at 1 h, whereas the peak of fra-1 induction occurred between 1 and 2 h after application of stretch. Treatment of myometrial cells with progesterone (100 nM, 400 nM, 1 microM) for 1 or 6 h before the application of static stretch did not affect the magnitude of the c-fos response. However, 24 h of progesterone exposure reduced the magnitude of c-fos and fosB stretch induction at both the 400 nM and 1 microM doses. These data indicate that several members of the AP-1 family are stretch-responsive genes in myometrial smooth muscle cells. This response can be attenuated by pretreatment with progesterone; however, the requirement for longer pretreatment times suggests that the inhibitory actions of progesterone do not occur through a direct action of the progesterone receptor within the promoter regions of AP-1 genes.     

Suppression of prostaglandin E(2)-mediated c-fos mRNA induction by interleukin-4 in murine macrophages

When murine peritoneal macrophages were stimulated for 30 min with arachidonic acid, the growth-associated immediate early gene c-fos was induced in a concentration-dependent manner as assessed by Northern blot analysis. The arachidonic acid-induced c-fos mRNA expression was inhibited by a cyclooxygenase inhibitor, indomethacin, but not by a lipoxygenase inhibitor, nordihydroguaiaretic acid. Macrophages produced prostaglandin (PG) E(2) from arachidonic acid as determined by an enzyme immunoassay. Northern blot analysis revealed the expression of PGE receptor EP2 and EP4 subtypes, but not EP1 and EP3 in murine macrophages. PGE(2) brought about a marked elevation of cAMP, and c-fos mRNA expression was increased by PGE(2) and dibutyryl cAMP in these cells. These results suggest that arachidonic acid is transformed to PGE(2), which then binds to EP2 and EP4 receptors to increase intracellular cAMP and c-fos mRNA expression. Furthermore, the induction of c-fos by arachidonic acid, PGE(2), and cAMP was suppressed by pretreatment with interleukin (IL)-4. We also showed that the tyrosine phosphorylation of a Janus kinase, JAK3, is enhanced by IL-4 treatment, suggesting that the PGE(2)-mediated c-fos mRNA induction is inhibited by IL-4 through the tyrosine phosphorylation of JAK3.     

Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells

The involvement of H2O2 in cataract development has been established inboth human patients and animal models. At the molecular level H2O2 has beenobserved to cause damage to DNA, protein and lipid. To explore the oxidativestress response of the lens system at the gene expression level, we haveexamined the effects of H2O2 on the mRNA change of the proto-oncogenes,c-jun, c-fos and c-myc in a rabbit lens cell line, N/N1003A. H2O2 treatmentof the rabbit lens epithelial cells for 60 min induces quick up-regulationof both c-jun and c-fos mRNAs. The maximal induction is 38 fold for c-jun at150 µM and 72 fold for c-fos at 250 µM H2O2. Treatment ofN/N1003A cells with 50-250 µM H2O2 for 60 min leads to a 2-5 foldincrease of the c-myc mRNA level. H2O2 also induces an up-regulation intransactivity of the activating protein-1 (AP-1) as shown with a reportergene driven by a prolactin gene promoter with 4 copies of AP-1 binding sitesinserted in the upstream of the promoter. Maximal induction occurs with 150µM H2O2. In the same system, the antioxidants, N-acetyl-cysteine (NAC)and pyrrolidine dithiocarbamate (PDTC) at concentrations shown toup-regulate the mRNAs of both c-jun and c-fos, also enhance thetransactivity of AP-1. NAC and PDTC have different effects in modulating theinduction of AP-1 activity by H2O2 and TPA. These results reveal thatoxidative stress regulates expression of various regulatory genes in lenssystems, which likely affects cell proliferation, differentiation andviability and thus affect normal lens functions.