Differential expression of immediate early genes in the hippocampus and spinal cord

We have demonstrated that immediate early genes can be differentially activated within the central nervous system. We examined the effects of tetanic stimulation in the hippocampus and of noxious sensory stimulation of the spinal cord on the expression of eight immediate early genes. Induction of long-term potentiation (LTP) in the dentate gyrus resulted in an increase in mRNA and protein for NGFI-A (also termed Zif/268, Egr-1, or Krox 24), and less consistently for jun-B mRNA. No increase was seen for c-fos, NGFI-B, c-jun, jun-D, SRF, or PC4 mRNAs. Blockade of the NMDA receptor prevented the induction of both LTP and NGFI-A mRNA in the dentate gyrus. However, commissural stimulation, which prevented the induction of LTP, resulted in bilateral activation of all the genes examined, including NGFI-A. No change was seen in animals trained in a water maze. These results suggest that no simple relationship exists between LTP, spatial learning, and immediate early gene induction. Stimulation of sensory fibers resulted in an increase in mRNA for NGFI-A, c-fos, SRF, NGFI-B, and c-jun in spinal cord neurons. Blockade of the NMDA receptor had no effect on immediate early gene induction in the spinal cord.     

Phospholipase C-gamma1 is required for the induction of immediate early genes by platelet-derived growth factor

To explore the functional role of phospholipase C-gamma1 (PLC-gamma1) in the induction of immediate early genes (IEGs), we have examined the influence of Plcg1 gene disruption on the expression of 14 IEG mRNAs induced by platelet-derived growth factor (PDGF). Plcg1-null embryos were used to produce immortalized fibroblasts genetically deficient in PLC-gamma1 (Null cells), and retroviral infection of those cells was used to derive PLC-gamma1 re-expressing cells (Null+ cells). In terms of PDGF activation of PDGF receptor tyrosine phosphorylation as well as the mitogen-activated protein kinases Erk1 and Erk2, Null and Null+ cells responded equivalently. However, the PDGF-dependent expression of all IEG mRNAs was diminished in cells lacking PLC-gamma1. The expression of FIC, COX-2, KC, JE, and c-fos mRNAs were most strongly compromised, as the stimulation of these genes was reduced by more than 90% in cells lacking PLC-gamma1. The combination of PMA and ionomycin, downstream analogs of PLC activation, did provoke expression of mRNAs for these IEGs in the Null cells. We conclude that PLC-gamma1 is necessary for the maximal expression of many PDGF-induced IEGs and is essential for significant induction of at least five IEGs.     

Interleukin-1-inducible tumor growth arrest is characterized by activation of cell type-specific "early" gene expression programs.

Human melanoma cells, A375-C6, were "committed" to growth arrest within a few hours of exposure to interleukin-1 (IL-1). Co-treatment with actinomycin D rescued the cells from the "commitment," suggesting that "early" gene activation events may be crucial for growth arrest. To understand the mechanism of IL-1 action, we are studying early genes whose expression is induced by the cytokine. Five early genes associated with IL-1 action in the melanoma cells were isolated by differential screening of a cDNA library, which was enriched for sequences representing IL-1 responsive genes (IRGs). Nucleotide sequencing identified four of the genes as gro-alpha, gro-beta, c-jun and nur77/NGF1-B/NAK1, respectively, while the fifth was judged as novel by GenBank search and designated IRG-9. None of the early genes was uniquely associated with the antiproliferative action of IL-1: other growth-inhibitory as well as growth-stimulatory signals induced these genes in diverse cell types. However, analysis of the induction patterns of the IRGs and other well known early genes revealed that IL-1 action in the melanoma cells is characterized by activation of a unique primary gene expression program. This program was defined by the magnitude and temporal pattern of induction of the five IRGs, feeble induction of c-fos, and lack of induction of Egr-1 and c-myc. We present evidence that this program is growth arrest-specific in the melanoma cells and that distinct cell type-specific programs are associated with IL-1 growth-regulatory actions in other tumor cells. Based on these data, we propose that early genes may play multifunctional roles in tumor growth control, but specificity for the growth arrest action of IL-1 is determined by the composite early gene induction program.     

Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen

Early in neonatal development, differentiated myocardial cells lose their ability to proliferate, and further enlargement of the heart occurs through hypertrophy of existing cardiac muscle cells. To study the process of myocardial growth and hypertrophy we have recently utilized a neonatal rat myocardial cell model (Lee, H. R., Henderson, S. A., Reynolds, R., Dunnmon, P., Yuan, D., and Chien, K. R. (1988) J. Biol. Chem. 263, 7352-7538). The present study was designed to determine if the expression of SV40 large T antigen would be capable of restoring the proliferative capacity of terminally differentiated neonatal rat myocardial cells. Utilizing a replication-defective recombinant human adenovirus which contains an SV40 early T antigen insert, maximal expression of T antigen was achieved at 24-48 h postinfection, with over 85-90% of the cells displaying positive T antigen staining. Furthermore, the expression of the T antigen-induced proliferation of the myocardial cells without the loss of expression of certain differentiated properties, including myosin light chain expression and assembly into organized myofibrils, spontaneous contractile activity, and a chronotropic response to adrenergic agonists. These results demonstrate the utility of recombinant human adenoviruses to achieve high efficiency transient expression of foreign genes in differentiated myocardial cells and suggest that the expression of T antigen may provide a suitable model to study the biochemical events which are required to maintain the proliferative capacity of myocardial cells.     

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0. 05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.     

Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy.

Although it is a well-known fact that hemodynamic load is a major determinant of cardiac muscle mass and its phenotype, little is known as to how mechanical load is converted into intracellular signals of gene regulation. To address this question, we characterized the stretch-induced adaptation of cultured neonatal cardiocytes grown on a stretchable substrate in a serum-free medium. Static stretch (20%) of the cells was applied without cell injury. Stretch caused hypertrophy in myocytes and hyperplasia in non-myocytes. Stretch caused an induction of immediate-early genes such as c-fos, c-jun, c-myc, JE, and Egr-1, but not Hsp70. Immunostaining showed that the stretch-induced Fos protein localized in the nucleus of both myocytes and non-myocytes. Nuclear extracts from stretched myocytes contained DNA binding activity to the AP-1 and Egr-1 consensus sequences. In myocytes, the induction of immediate-early genes was followed by expression of "fetal" genes such as skeletal alpha-actin, atrial natriuretic factor, and beta-myosin heavy chain. DNA transfection experiments showed that the "stretch-response element" of the c-fos gene promoter is present within 356 base pairs of the 5'-flanking region, whereas that of the atrial natriuretic factor and the beta-myosin heavy chain genes is probably located outside of 3412 and 628 base pairs of the 5'-flanking region, respectively. These results demonstrate that the phenotype of stretched cardiocytes in this in vitro model closely mimics that of hemodynamic load-induced hypertrophy in vivo. This model seems to be a suitable system with which to dissect the molecular mechanisms of load-induced hypertrophy of cardiac muscle.     

Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.