Induction Of Immediate-early Response Genes Research Articles

Fibroblast growth factor 15 deficiency impairs liver regeneration in mice

Fibroblast growth factor (FGF) 15 (human homolog, FGF19) is an endocrine FGF highly expressed in the small intestine of mice. Emerging evidence suggests that FGF15 is critical for regulating hepatic functions; however, the role of FGF15 in liver regeneration is unclear. This study assessed whether liver regeneration is altered in FGF15 knockout (KO) mice following 2/3 partial hepatectomy (PHx). The results showed that FGF15 KO mice had marked mortality, with the survival rate influenced by genetic background. Compared with wild-type mice, the KO mice displayed extensive liver necrosis and marked elevation of serum bile acids and bilirubin. Furthermore, hepatocyte proliferation was reduced in the KO mice because of impaired cell cycle progression. After PHx, the KO mice had weaker activation of signaling pathways that are important for liver regeneration, including signal transducer and activator of transcription 3, nuclear factor-κB, and mitogen-activated protein kinase. Examination of the KO mice at early time points after PHx revealed a reduced and/or delayed induction of immediate-early response genes, including growth-control transcription factors that are critical for liver regeneration. In conclusion, the results suggest that FGF15 deficiency severely impairs liver regeneration in mice after PHx. The underlying mechanism is likely the result of disrupted bile acid homeostasis and impaired priming of hepatocyte proliferation.

The translation inhibitor anisomycin induces Elk-1-mediated transcriptional activation of egr-1 through multiple mitogen-activated protein kinase pathways

The early growth response-1 gene (egr-1) encodes a zinc-finger transcription factor Egr-1 and is rapidly inducible by a variety of extracellular stimuli. Anisomycin (ANX), a protein synthesis inhibitor, stimulates mitogen-activated protein kinase (MAPK) pathways and thereby causes a rapid induction of immediate-early response genes. We found that anisomycin treatment of U87MG glioma cells resulted in a marked, time-dependent increase in levels of Egr-1 protein. The results of Northern blot analysis and reporter gene assay of egr-1 gene promoter (Pegr-1) activity indicate that the ANX- induced increase in Egr-1 occurs at the transcriptional level. Deletion of the serum response element (SRE) in the 5'-flanking region of egr-1 gene abolished ANX-induced Pegr-1 activity. ANX induced the phosphorylation of the ERK1/2, JNK, and p38 MAPKs in a time-dependent manner and also induced transactivation of Gal4-Elk-1, suggesting that Elk-1 is involved in SRE-mediated egr-1 transcription. Transient transfection of dominant-negative constructs of MAPK pathways blocked ANX-induced Pegr-1 activity. Furthermore, pretreatment with specific MAPK pathway inhibitors, including the MEK inhibitor U0126, the JNK inhibitor SP600125, and the p38 kinase inhibitor SB202190, completely inhibited ANX-inducible expression of Egr-1. Taken together, these results suggest that all three MAPK pathways play a crucial role in ANX-induced transcriptional activation of Pegr-1 through SRE-mediated transactivation of Elk-1.

The regulation of normal melanocyte proliferation.

In vitro, normal human melanocytes require synergistic mitogens, in addition to the common growth factors present in serum, in order to proliferate. The peptide growth factors that confer stimulation are fibroblast growth factors (such as bFGF/FGF2), hepatocyte growth factor/scatter factor (HGF/SF), mast/stem cell factor (M/SCF), endothelins (such as ET-1) and melanotropin (MSH). The proper function of these factors and their cognate receptors is likely to be important in vivo, as all five ligands are produced in the skin, and disruption of their normal function, by elimination due to deletions or mutations, or overproduction due to ectopic expression, disrupts the normal distribution of melanocytes. The synergistic growth factors activate intracellular signal transduction cascades and maintain the intermediate effectors at optimal levels and duration required for nuclear translocation and modification of transcription factors. The consequent induction of immediate-early response genes, such as cyclins, and subsequent activation of cyclin-dependent kinases (CDK4, CDK6 and CDK2) inactivates the retinoblastoma family of proteins (pRb, p107 and p130, together termed pocket proteins), and releases their suppressive association with E2F transcription factors. Molecular events that disrupt this tight control of pocket proteins and cause their inactivation, increase E2F transcriptional activity and confer autonomous growth on melanocytes.

Role of the STAT1-SH2 Domain and STAT2 in the Activation and Nuclear Translocation of STAT1

Interferon (IFN) induction of immediate-early response genes is mediated through the signal transducers and activators of transcription (STATs). Activation of STAT1 by IFNalpha or IFNgamma through its tyrosine phosphorylation involves members of the Jak tyrosine kinases. In addition, STAT2 is activated by IFNalpha, and, together with STAT1 and p48/ISGF3gamma, forms the transcription factor complex ISGF3. Previous findings suggested that the STAT1-SH2 domain, which is required for the homo- or heterodimerization of STAT1, also participates in the recruitment of STAT1 to the IFN-receptors, because mutations in the SH2-domain abolished STAT1 activation by IFNgamma. Furthermore, STAT2 was reported to be required for the activation of STAT1 by IFNalpha. We were able to induce STAT1 tyrosine phosphorylation by IFNalpha/beta in the absence of STAT2 or a functional STAT1-SH2 domain. In contrast, IFNgamma was unable to cause tyrosine phosphorylation of STAT1-(SH2:Arg --> Gln). Interestingly, although STAT1 was found in the nucleus in STAT2-deficient cells, the nuclear accumulation of the tyrosine phosphorylated SH2-mutant STAT1 was impaired. In summary, our results indicate that the SH2 domain of STAT1 is not required for its ligand-dependent activation by IFNalpha/beta. Moreover, tyrosine phosphorylation is not sufficient to target STAT1 to the nucleus; rather, dimerization appears to play a critical role in the subcellular distribution of STAT1.

Glucose and glucoincretin peptides synergize to induce c‐fos, c‐jun,junB,zif‐268, and nur‐77gene expression in pancreatic β(INS‐1) cells

To link glucose signaling to its long-term pleiotropic effects in the pancreatic beta-cell, we have investigated whether glucose regulates immediate-early response genes (IEGs) coding for transcription factors implicated in cell proliferation and differentiation. Glucose causes a coordinated transcriptional activation of the IEGs c-fos, c-jun, JunB, zif-268, and nur-77 in the pancreatic beta-cell line INS-1. This activation is entirely dependent on the presence of the cell-permeant cAMP analog chlorophenylthio-cAMP, which has only a modest effect by itself. The accumulation of c-fos, JunB, and nur-77 mRNA occurs at physiological concentrations of glucose (3 to 11 mM), requires a 1-2 h period, and is mimicked by other nutrient stimuli including mannose, leucine plus glutamine, and pyruvate. Glucose is synergistic with the glucoincretin peptides GLP-1 and PACAP-38, whereas these neurohormonal agents have no effect at low (3 mM) glucose. Mechanistically, the synergy between glucose and the glucoincretins is not based on cAMP alone as glucose does not further increase intracellular cAMP in response to GLP-1 and PACAP-38. A role for Ca2+ signaling is inferred, since the L-type Ca2+ channel blocker nifedipine markedly reduces the induction of c-fos and nur-77 by glucose and GLP-1. The induction of IEGs by glucose and chlorophenylthio-cAMP or GLP-1 and the inhibitory effect of nifedipine are also observed in the betaHC9 cell line. The results indicate that GLP-1 and PACAP-38 act as competence factors for the action of glucose on c-fos, JunB, and nur-77. It is suggested that the synergistic effect of glucose and glucoincretins on IEG expression plays an important role in the adaptive processes of the beta-cell to hyperglycemia.

Maternal Deprivation and Stress Induce Immediate Early Genes in the Infant Rat Brain

The hypothalamic-pituitary-adrenal (HPA) axis is normally quiescent during the stress-hyporesponsive period (SHRP) from day 4–14 in infant rats. However, maternal deprivation (DEP) can disinhibit the HPA axis, thus enabling neonatal rats to respond to mild stressors. In an effort to understand how DEP may alter HPA axis sensitivity, we used in situ hybridization to measure changes in the expression of stress-responsive genes in the brains of neonatal rats. Despite the minimal HPA axis response in nondeprived rats during the SHRP (postnatal day 12), the mild stress of a saline injection significantly increased messenger RNA levels of two immediate-early genes (IEGs), c-fos and NGFI-B, in the hypothalamic paraventricular nucleus (PVN) and in the cerebral cortex. Following 24 h of DEP, the induction of IEGs in response to stress was greatly potentiated in the PVN of P12 neonates. In contrast, DEP attenuated the effects of stress on IEG induction in rats that had matured beyond the SHRP (P20). Surprisingly, DEP decreased basal levels of CRH messenger RNA in the PVN at P12 and P20. Thus the SHRP most accurately refers to HPA axis insensitivity to stress because the brain itself readily responds to stress as evidenced by the induction of IEGs.

Maternal deprivation and stress induce immediate early genes in the infant rat brain.

The hypothalamic-pituitary-adrenal (HPA) axis is normally quiescent during the stress-hyporesponsive period (SHRP) from day 4-14 in infant rats. However, maternal deprivation (DEP) can disinhibit the HPA axis, thus enabling neonatal rats to respond to mild stressors. In an effort to understand how DEP may alter HPA axis sensitivity, we used in situ hybridization to measure changes in the expression of stress-responsive genes in the brains of neonatal rats. Despite the minimal HPA axis response in nondeprived rats during the SHRP (postnatal day 12), the mild stress of a saline injection significantly increased messenger RNA levels of two immediate-early genes (IEGs), c-fos and NGFI-B, in the hypothalamic paraventricular nucleus (PVN) and in the cerebral cortex. Following 24 h of DEP, the induction of IEGs in response to stress was greatly potentiated in the PVN of P12 neonates. In contrast, DEP attenuated the effects of stress on IEG induction in rats that had matured beyond the SHRP (P20). Surprisingly, DEP decreased basal levels of CRH messenger RNA in the PVN at P12 and P20. Thus the SHRP most accurately refers to HPA axis insensitivity to stress because the brain itself readily responds to stress as evidenced by the induction of IEGs.

Identification of Upstream Signals Regulating Interleukin-6 Gene Expression During In Vitro Treatment of Human B Cells With Pokeweed Mitogen

The present studies have characterized the regulation of interleukin-6 (IL-6) gene expression during pokeweed mitogen (PWM)-driven human B-cell differentiation. PWM induced an early and transient increase in the expression of immediate-early response genes of the jun/fos leucine zipper family (c-jun, jun B, c-fos, and fos-B). The induction of c-jun mRNA by PWM was concentration dependent. Nuclear run-on assays showed that PWM treatment is associated with an increased rate of c-jun gene transcription. The induction of c-jun mRNA precedes the induction of IL-6 gene expression and IL-6 secretion by the B cells. c-Jun antisense, but not sense, oligodeoxynucleotide (ODN) significantly decreases PWM-related B-cell (1) proliferation; (2) IL-6 mRNA induction; (3) IL-6 secretion; and (4) nuclear extract binding to AP-1 in electrophoretic mobility shift assay. In contrast, c-Fos anti-sense ODN did not effect either IL-6 mRNA induction or IL-6 secretion triggered in B cells by PWM. The results further show activation of c-Raf-1 kinase in PWM-treated B cells. Raf-1 acts upstream to mitogen-activated protein (MAP) kinase; therefore, studies were performed to assay for MAP kinase activation in these cells. The results show an increase in phosphorylation of myelin basic protein (MBP) and c-Jun "Y" peptide in PWM-treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signaling cascade in normal human splenic B cells, which, at least in part, involves serine/threonine protein kinases. These results show transient induction of immediate-early response genes in B cells and support a potential role for the c-jun gene product in regulation of IL-6 transcription and secretion.

PMA Inhibits the Growth of Human Fibroblasts after the Induction of Immediate-Early Genes

WS-1 cells, human fibroblasts derived from embryonic skin, become quiescent in serum-free medium. Addition of serum or thrombin stimulates incorporation of 3 H-labeled thymidine 16-40 h later. The tumor promoter phorbol 12-myristate 13-acetate (PMA) inhibited this response in quiescent cells stimulated with serum or thrombin. PMA also inhibited thymidine incorporation in rapidly growing WS-1 cells. Since the inhibition caused by PMA could result from activation of protein kinase C (PKC) or the down-regulation of PKC, the effect of the PKC inhibitor, GF 109203X, was tested. GF 109203X did not affect thymidine incorporation induced by serum. GF 109203X prevented the inhibition caused by PMA, suggesting that the activation of PKC is required for the inhibition of DNA synthesis. A prolonged activation of PKC is probably required, since a single treatment of a synthetic diacylglycerol does not inhibit and addition of GF 109203X 2 to 8 h after PMA reverses the inhibition. One possible target for PMA activity is the induction of immediate-early response genes. Thrombin or serum treatment of quiescent WS-1 cells induced protooncogenes c- fos and c- jun. PMA also induced c- fos and c- jun with a time course similar to that of serum or thrombin. The induction of c- fos by PMA was sensitive to staurosporine or GF 109203X. A time course of PMA addition indicated that maximal inhibition of DNA synthesis occurred when PMA was added 4 h after stimulation with serum. These results suggest that the PMA-induced inhibition of DNA synthesis occurs through an activation of PKC that inhibits DNA synthesis at a point after the induction of c- fos and c- jun.

Differential effects of rabies and borna disease viruses on immediate-early- and late-response gene expression in brain tissues

In situ hybridization and Northern blot analysis were used to examine expression of the immediate-early-response genes (IEGs) egr-1, junB, and c-fos, and the late response gene encoding enkephalin in the brains of rats infected intranasally with Borna disease virus (BDV) or rabies virus. In both Borna disease and rabies virus infections, a dramatic and specific induction of IEGs was detected in particular regions of the hippocampus and the cortex. Increased IEG mRNA expression overlapped with the characteristic expression patterns of BDV RNA and rabies virus RNA, although relative expression levels of viral RNA and IEG mRNA differed, particularly in the hippocampal formation. Furthermore, the temporal relationship between viral RNA synthesis and activation of IEG mRNA expression in BDV infection differed markedly from that in rabies virus infection, suggesting that IEG expression is upregulated by different mechanisms. Expression of proenkephalin (pENK) mRNA was also significantly increased in BDV infection, whereas in rabies virus infection, pENK mRNA levels and also the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA were reduced at terminal stages of the disease, probably reflecting a generalized suppression of cellular protein synthesis due to massive production of rabies virus mRNA. The correlation between activated IEG mRNA expression and the strong increase in viral RNA raises the possibility that IEG products induce some phenotypic changes in neurons that render them more susceptible to viral replication.

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.