Intrinsic Negative Regulator Research Articles

Inhibitors of p38 mitogen‐activated protein kinase enhance proliferation of mouse neural stem cells

The p38 mitogen-activated protein kinase (MAPK) is induced in response to environmental stress. Although p38 MAPK has been implicated in diverse cellular processes, including cell proliferation, differentiation, and survival of differentiated cells in the central nervous system (CNS), the expression profile and roles of p38 MAPK in the developing brain remain largely unknown. In the present study, we demonstrate that p38 MAPK is expressed predominantly in nestin-positive cells in the cerebral cortex in embryonic day 10 (E10) brain and that expression of the protein decreases gradually during development. To investigate the roles of p38 MAPK in the embryonic brain, two selective p38 MAPK inhibitors, SB202190 and SB203580, were added to the primary neuronal cultures from E10-E14 brains. After 7 days of exposure to these inhibitors, but not SB202474, a negative analog of SB203580, numerous large neurospheres were present. MAPK inhibitors also selectively increased the growth rate of neural stem cells (NSCs) purified from secondary neurospheres and the number of bromodeoxyuridine-positive NSCs. Thus, p38 MAPK inhibitors are potent stimulators of NSC proliferation, and p38 MAPK may be an intrinsic negative regulator of NSC proliferation during early brain development.

Intrinsic Negative Cell Cycle Regulation Provided by PIP Box- and Cul4Cdt2-Mediated Destruction of E2f1 during S Phase

Intrinsic Negative Cell Cycle Regulation Provided by PIP Box- and Cul4Cdt2-Mediated Destruction of E2f1 during S Phase

Abstract 911: Lacking SOCS3 Gene in Macrophages Prevents the Development of Atherosclerosis in ApoE Deficient Mice

Background : Atherosclerosis is now generally accepted as a chronic inflammatory disease characterized by accumulation of blood-derived macrophages in the arterial wall. It has been shown that suppressor of cytokine signaling 3 (SOCS3) is an intrinsic negative regulator of interleukin-6 (IL-6)-STAT3 signaling that plays an essential role in inflammatory response. In the present study, we determined whether STAT3-mediated IL-6 signaling and SOCS3 within the macrophages would play an important role in the development of atherosclerosis. Methods and Results : We examined the activation of STAT3 and expressions of IL-6 family cytokines and SOCS3 in the aorta of apolipoprotein E knockout mice (ApoE-KO) and control mice by western blot, immunohistochemical staining and RT-PCR. Western blot revealed that STAT3 is phosphorylated in the atherosclerotic lesions from ApoE-KO mice but not in those from control mice. Dual immunohistochemical staining revealed that phosphorylated STAT3 and macrophage marker, MOMA2, were co-expressed in the atherosclerotic plaque in ApoE-KO deficient mice, suggesting that the STAT3 signaling pathway within the macrophages was activated in atherosclerosis. Next, to investigate the role of STAT3-mediated IL-6 signaling and SOCS3 in macrophages during atherogenesis, we created ApoE-KO mice with a macrophage-restricted deletion of SOCS3 gene (ApoE/SOCS3-KO). We found that atherosclerotic lesion area by oil-red O staining was significantly reduced in ApoE/SOCS3-KO (n=17) compared with control ApoE-KO mice (n=13) (4.63±0.43 vs. 8.29±0.50 %, p<0.01). In in vitro analyses, enzyme-linked immunosorbent assay revealed that LPS-induced TNF-alpha production were significantly smaller in SOCS3 deficient macrophages compared with wild type macrophages (n=8, 2238±155 vs. 992±83 pg/ml, p<0.01). Also we found that IL-6-induced oxidized-LDL uptake was 42% reduced in SOCS3 deficient macrophages compared with wild type macrophages (p<0.01). Conclusion: Our data show that the deletion of SOCS3 in macrophages prevents the development of atherosclerosis in mice, possibly by inhibiting the inflammatory cytokine production and oxidized-LDL uptake in macrophages.

TRAF6 is a T cell–intrinsic negative regulator required for the maintenance of immune homeostasis

TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell-specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4+ CD25+ regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell-intrinsic control mechanism to render responder T cells susceptible to tolerizing signals.

S100B Expression Modulates Left Ventricular Remodeling After Myocardial Infarction in Mice

S100B, a 20-kDa, Ca2+-binding dimer, is a putative intrinsic negative regulator of myocardial hypertrophy expressed after myocardial infarction. S100B-overexpressing transgenic (TG) and S100B-knockout (KO) mice have been generated to assess the consequences of S100B expression and altered hypertrophy after infarction. We compared 21 wild-type (WT), 20 TG, and 24 KO mice over 35 days after experimental myocardial infarction with sham-operated controls (n=56). Of those, 4 WT-infarcted mice, 7 TG-infarcted mice, and 1 KO-infarcted mouse and no sham-operated mice died during the observation period. Among survivors, echocardiography, hemodynamic studies, and postmortem examination indicated that the WT and KO groups of infarcted mice mounted a hypertrophic response that was augmented in KO mice. The S100B-overexpressing TG group did not develop hypertrophy but demonstrated increased apoptosis. The postinfarct end-diastolic pressure was lower in KO mice than in WT mice, in accordance with other structural, hemodynamic, and functional parameters, which suggests that abrogation of S100B expression augmented hypertrophy, decreased apoptosis, and was beneficial to preservation of cardiac function within this time frame. S100B regulates the hypertrophic response and remodeling in the early postinfarct period and represents a potential novel therapeutic target.

Interferon-α stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection

RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore plays a central role in the hepatitis-delta-virus (HDV) life-cycle. Editing is catalyzed by the enzyme Adenosine-deaminase-acting-on-RNA1 (ADAR1) of which two different forms, ADAR1-L and ADAR1-S, exist. As ADAR1-L is induced by interferon (IFN)-alpha, we examined the influence of IFN-alpha-stimulation of host cells on HDV-RNA editing. Editing was studied in Huh-7-cells transfected with HDV-RNA on days 7, 14, 21 and 28 after transfection. ADAR1-L mRNA was measured by RT-PCR. IFN-alpha-treatment led to a 5-fold higher expression of ADAR1-L and to an increase in editing from 14+/-2% (SD) in unstimulated controls to 27+/-4% (SD) on day 7 after transfection. Editing further increases over time to the same maximum level of 35% in IFN-alpha-treated as well as untreated cells. By IFN-alpha-stimulation both ADAR1-L expression and editing are increased in Huh-7-cells at day 7, and the maximum level of edited antigenomes is reached earlier with IFN-alpha-treatment as compared to untreated cells. Thus, ADAR1-L appears to be able to increase editing, but the HDV genome apparently has an intrinsic negative feed-back regulation mechanism that limits editing to roughly a third of the genomes.

Inhibition of norepinephrine-induced cardiac hypertrophy in s100beta transgenic mice.

We have recently reported that the Ca2+-binding protein S100beta was induced in rat heart after infarction and forced expression of S100beta in neonatal rat cardiac myocyte cultures inhibited alpha1-adrenergic induction of beta myosin heavy chain (MHC) and skeletal alpha-actin (skACT). We now extend this work by showing that S100beta is induced in hearts of human subjects after myocardial infarction. Furthermore, to determine whether overexpression of S100beta was sufficient to inhibit in vivo hypertrophy, transgenic mice containing multiple copies of the human gene under the control of its own promoter, and CD1 control mice were treated with norepinephrine (NE) (1.5 mg/kg) or vehicle, intraperitoneally twice daily for 15 d. In CD1, NE produced an increase in left ventricular/body weight ratio, ventricular wall thickness, induction of skACT, atrial natriuretic factor, betaMHC, and downregulation of alphaMHC. In transgenic mice, NE induced S100beta transgene mRNA and protein, but provoked neither hypertrophy nor regulated cardiac-specific gene expression. NE induced hypertrophy in cultured CD1 but not S100beta transgenic myocytes, confirming that the effects of S100beta on cardiac mass reflected myocyte-specific responses. These transgenic studies complement in vitro data and support the hypothesis that S100beta acts as an intrinsic negative regulator of the myocardial hypertrophic response.

S100β Inhibits α1-Adrenergic Induction of the Hypertrophic Phenotype in Cardiac Myocytes

In an experimental rat model of myocardial infarction, surviving cardiac myocytes undergo hypertrophy in response to trophic effectors. This response involves gene reprogramming manifested by the re-expression of fetal genes, such as the previously reported isoform switch from adult alpha- to embryonic beta-myosin heavy chain. We now report the transient re-expression of a second fetal gene, skeletal alpha-actin in rat myocardium at 7 days post-infarction, and its subsequent down-regulation coincident with the delayed induction of S100beta, a protein normally expressed in brain. In cultured neonatal rat cardiac myocytes, co-transfection with an S100beta-expression vector inhibits a pathway associated with hypertrophy, namely, alpha1-adrenergic induction of beta-myosin heavy chain and skeletal alpha-actin promoters mediated by beta-protein kinase C. The induction of beta-myosin heavy chain by hypoxia was similarly blocked by forced expression of S100beta. Our results suggest that S100beta may be an intrinsic negative regulator of the hypertrophic response of surviving cardiac myocytes post-infarction. Such negative regulators may be important in limiting the adverse consequences of unchecked hypertrophy leading to ventricular remodeling and dysfunction.

Role of CBP/P300 in nuclear receptor signalling.

The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.

Purification of recombinant [formula omitted] proteins of phospholipase C-γ1 and -γ2 and their inhibitory effect on PIP 2-hydrolysis induced by both types of phospholipase C-γ

In order to examine physiological function of the SH2 SH3 region of phospholipase C-γ (Z region), we independently expressed cDNA fragments corresponding to the SH2 SH3 region of PLC-γ1 and PLC-γ2 in Escherichia coli. Although these recombinant proteins were recovered in particulate fractions by centrifugation of cell extracts, they were successfully solubilized by guanidium hydrochloride and then purified to homogeneity by heparin column chromatography. The molecular mass of the proteins was 45 kDa (derived from PLC-γ1 and designated as rP45Z) and 38 kDa (derived from PLC-γ2 and designated as rP38Z), which was consistent with that as expected from inserted cDNA. We determined the effect of purified rP45Z or rP38Z on PIP 2-hydrolyzing activity of either PLC-γ1 or PLC-γ2 and found that these proteins strongly suppressed the rate of PLC-dependent PIP 2-hydrolysis. Furthermore, both rP45Z and rP38Z were phosphorylated at tyrosine residue by epidermal growth factor receptors and their inhibitory effect on PIP 2-hydrolysis was significantly decreased by this phosphorylation. These results indicate that the Z region might be involved in autoregulation of PLC-γ as intrinsic negative regulator.