Autoregulatory Feedback Research Articles

Nuclear Calpain Regulates Ca2+-dependent Signaling via Proteolysis of Nuclear Ca2+/Calmodulin-dependent Protein Kinase Type IV in Cultured Neurons

Accumulating evidence indicates that calpains can reside in or translocate to the cell nucleus, but their functions in this compartment remain poorly understood. Dissociated cultures of cerebellar granule cells (GCs) demonstrate improved long-term survival when their growth medium is supplemented with depolarizing agents that stimulate Ca(2+) influx and activate calmodulin-dependent signaling cascades, notably 20 mm KCl. We previously observed Ca(2+)-dependent down-regulation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) type IV, which was attenuated by calpain inhibitors, in GCs supplemented with 20 mm KCl (Tremper-Wells, B., Mathur, A., Beaman-Hall, C. M., and Vallano, M. L. (2002) J. Neurochem. 81, 314-324). CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival. Here, we demonstrate by immunolocalization/confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mm KCl. Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and non-nuclear calpain substrates were not degraded under chronic depolarizing culture conditions. Depolarization and Ca(2+)-dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca(2+)-CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by approximately 10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced. We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca(2+)-CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mm KCl. This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca(2+) influx.

A Novel Mouse Model of Hypogonadotrophic Hypogonadism: N-Ethyl-N-Nitrosourea-Induced Gonadotropin-Releasing Hormone Receptor Gene Mutation

An autosomal-recessive mutation that causes hypogonadotrophic hypogonadism was isolated during an N-ethyl-N-nitrosourea mutagenesis screen in mice. Affected males had micropenis and small, undescended testes with spermatogenesis arrested at the pachytene stage of meiosis, leading to sterility. Androgen-sensitive organs were small and immature. Affected females were externally normal but sterile with small ovaries due to an arrest at the secondary stage of folliculogenesis, and the uterus and oviducts were thin and immature. Circulating reproductive hormones were significantly decreased in affected males and females. There was also a dramatic reduction in the numbers of FSH- and LH-producing gonadotrophs. Meiotic mapping of the mutation and candidate gene sequencing determined that the N-ethyl-N-nitrosourea-induced lesion is in the third transmembrane domain of the GnRH receptor gene (Gnrhr). In vitro studies indicate that the mutant receptor is not coupled to the plasma membrane signal transduction system. Moreover, this mutant cannot be rescued with defined GnRH receptor pharmacoperones (pharmacological chaperones), an approach that rescues many other misfolded mutants. The mutant GnRH receptor was also shown to exert a dominant-negative effect on wild-type receptor function, indicating that the mutant receptor is unable to fold properly and likely misrouted within the cell, not reaching the plasma membrane. Surprisingly, Gnrhr mutant transcripts were significantly up-regulated in the pituitaries of Gnrhr mutants, revealing a previously unknown autoregulatory feedback loop. This is the first report of a mouse with a Gnrhr loss of function mutation. These GnRH-insensitive mice provide a novel animal model for the study of human idiopathic hypogonadotrophic hypogonadism.

Dapk1, encoding an activator of a p19ARF-p53-mediated apoptotic checkpoint, is a transcription target of p53

The p53 tumour suppressor functions as a transcriptional activator, and several p53-inducible genes that play a critical proapoptotic role have been described. Moreover, p53 regulates the expression of various proteins participating in autoregulatory feedback loops, including proteins that negatively control p53 stability (Mdm2 and Pirh2) or modulate stress-induced phosphorylation of p53 on Ser-46 (p53DINP1 or Wip1), a key event for p53-induced apoptosis. Here, we describe a new systematic analysis of p53 targets using oligonucleotide chips, and report the identification of dapk1 as a novel p53 target. We demonstrate that dapk1 mRNA levels increase in a p53-dependent manner in various cellular settings. Both human and mouse dapk1 genomic loci contain DNA sequences that bind p53 in vitro and in vivo. Since dapk1 encodes a serine/threonine kinase previously shown to suppress oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint, our results suggest that Dapk1 participates in a new positive feedback loop controlling p53 activation and apoptosis.

Feedback effects of circulating norepinephrine on sympathetic outflow in healthy subjects

The amplitude of low-frequency (LF) oscillations of heart rate (HR) usually reflects the magnitude of sympathetic activity, but during some conditions, e.g., physical exercise, high sympathetic activity results in a paradoxical decrease of LF oscillations of HR. We tested the hypothesis that this phenomenon may result from a feedback inhibition of sympathetic outflow caused by circulating norepinephrine (NE). A physiological dose of NE (100 ng.kg(-1).min(-1)) was infused into eight healthy subjects, and infusion was continued after alpha-adrenergic blockade [with phentolamine (Phe)]. Muscle sympathetic nervous activity (MSNA) from the peroneal nerve, LF (0.04-0.15 Hz) and high frequency (HF; 0.15-0.40 Hz) spectral components of HR variability, and systolic blood pressure variability were analyzed at baseline, during NE infusion, and during NE infusion after Phe administration. The NE infusion increased the mean blood pressure and decreased the average HR (P < 0.01 for both). MSNA (10 +/- 2 vs. 2 +/- 1 bursts/min, P < 0.01), LF oscillations of HR (43 +/- 13 vs. 35 +/- 13 normalized units, P < 0.05), and systolic blood pressure (3.1 +/- 2.3 vs. 2.0 +/- 1.1 mmHg2, P < 0.05) decreased significantly during the NE infusion. During the NE infusion after PHE, average HR and mean blood pressure returned to baseline levels. However, MSNA (4 +/- 2 bursts/min), LF power of HR (33 +/- 9 normalized units), and systolic blood pressure variability (1.7 +/- 1.1 mmHg2) remained significantly (P < 0.05 for all) below baseline values. Baroreflex gain did not change significantly during the interventions. Elevated levels of circulating NE cause a feedback inhibition on sympathetic outflow in healthy subjects. These inhibitory effects do not seem to be mediated by pressor effects on the baroreflex loop but perhaps by a presynaptic autoregulatory feedback mechanism or some other mechanism that is not prevented by a nonselective alpha-adrenergic blockade.

Structural analysis and identification of novel isoforms of the circadian clock gene period in the silk moth Bombyx mori.

The molecular basis of the circadian clock is an autoregulatory feedback loop in which the PAS domain-containing protein PERIOD periodically inhibits its own transcription. In the present study on PERIOD of the silk moth Bombyx mori, we have cloned two distinct period mRNA homologues with different PAS domain sequences either with or without the pentapeptide GTQEK. A period cDNA fragment first amplified by PCR exhibited a 15 bp-deleted nucleotide sequence in the PAS domain, compared with the database sequence. A possible alternative splicing mechanism was examined by PCR analyses, and a 15 bp-inserted clone was also amplified. The entire sequences of these period alpha and period beta isoforms were then determined by the 3' and 5' RACE methods. Isoform period alpha consists of a 3,324 bp oligonucleotide encoding 1,108 amino acid residues, whereas isoform period beta comprises 3,309 bp corresponding to 1,103 amino acids. Isoforms period alpha and period beta were found to be exactly identical except for the 15 bp deletion/insertion site. Such a pair of isoforms with a deletion/insertion sequence, namely two splice variants, has previously been reported only for the PERIOD proteins of the two honeybees, Apis mellifera and A. cerana. The occurrence of an alternative splicing mechanism in the B. mori period gene was hypothesized based on the genome structure recently clarified. Bombyx mori PERIOD alpha and beta proteins are the isomers that reveal firstly the different PAS domain sequences.

Gfi1:Green Fluorescent Protein Knock-in Mutant Reveals Differential Expression and Autoregulation of the Growth Factor Independence 1 (Gfi1) Gene during Lymphocyte Development

The Gfi1 gene encodes a 55-kDa transcriptional repressor protein with important functions in T-cell development, in granulopoiesis, and in the regulation of the innate immune response. To follow expression of the Gfi1 gene during the differentiation of specific immune cells, we have generated a mouse mutant in which the Gfi1 coding region is replaced by the gene for the green fluorescent protein (GFP). We found that Gfi1 gene expression is highest in early B-cell subpopulation and differentially expressed during T-cell development with peak levels at stages where pre-TCR or positive/negative selection takes place. Gfi1 is absent in mature B-cells, whereas in peripheral T-cells Gfi1 gene expression is low but rises significantly upon T-cell receptor triggering and decreases again in T-memory cells. Constitutive expression of an lck promoter-driven Gfi1 transgene led to transcriptional silencing of the Gfi1:GFP allele in T-cells. Because Gfi1 was found to occupy genomic sites of its own promoter in thymocytes and was able to repress its own transcription in vitro we propose that transcription of the Gfi1 gene is regulated through an autoregulatory feedback loop.

A nitrate-induced frq-less oscillator in Neurospora crassa.

When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq(9) mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the white-collar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.

NF-κB Is Essential for Induction of CYLD, the Negative Regulator of NF-κB: EVIDENCE FOR A NOVEL INDUCIBLE AUTOREGULATORY FEEDBACK PATHWAY

The transcription factor NF-kappaB regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-kappaB are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-kappaB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-kappaB essential modulator, also known as IkappaB kinase gamma). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-kappaB by TNF-alpha and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-kappaB signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-kappaB-dependent induction of CYLD by TNF-alpha and NTHi. These findings provide novel insights into the autoregulation of NF-kappaB activation.

Serine phosphorylation of mCRY1 and mCRY2 by mitogen-activated protein kinase.

The circadian oscillator is composed of a transcription/translation-based autoregulatory feedback loop in which Cryptochromes and Periods function as negative regulators for their own gene expression. Although post-translational modifications such as phosphorylation of these regulators appear crucial for circadian time-keeping mechanism, less is known about responsible protein kinases and their contribution to the function of the regulators. We found that mitogen-activated protein kinase (MAPK) associates with and phosphorylates mouse Cryptochromes (mCRY1 and mCRY2). Mass spectrometry analysis identified Ser265 and Ser557 of mCRY2 to be in vitro phospho-acceptor residues. Mutations of both the Ser residues to Ala completely abolished MAPK-mediated mCRY2 phosphorylation, suggesting that the two residues are the principal phosphorylation sites in mCRY2. Similarly, MAPK phosphorylates mCRY1 at Ser247, a site corresponding to Ser265 of mCRY2. An effect of the Ser phosphorylation was investigated by mutating Ser247 of mCRY1 and Ser265 of mCRY2 to Asp, which resulted in attenuation of each mCRYs' ability to inhibit BMAL1: CLOCK-mediated transcription, whereas a similar mutation at Ser557 of mCRY2 induced no measurable change in its activity. These results illustrate a model of MAPK-mediated negative regulation of mCRY function by phosphorylation at the specific Ser residue.

Platelet Factor XIII and Calpain Negatively Regulate Integrin αIIbβ3 Adhesive Function and Thrombus Growth

Excessive accumulation of platelets at sites of athero-sclerotic plaque rupture leads to the development of arterial thrombi, precipitating clinical events such as the acute coronary syndromes and ischemic stroke. The major platelet adhesion receptor glycoprotein (GP) IIb-IIIa (integrin alpha(IIb)beta3) plays a central role in this process by promoting platelet aggregation and thrombus formation. We demonstrate here a novel mechanism down-regulating integrin alpha(IIb)beta3 adhesive function, involving platelet factor XIII (FXIII) and calpain, which serves to limit platelet aggregate formation and thrombus growth. This mechanism principally occurs in collagen-adherent platelets and is induced by prolonged elevations in cytosolic calcium, leading to dramatic changes in platelet morphology (membrane contraction, fragmentation, and microvesiculation) and a specific reduction in integrin alpha(IIb)beta3 adhesive function. Adhesion receptor signal transduction plays a major role in the process by sustaining cytosolic calcium flux necessary for calpain and FXIII activation. Analysis of thrombus formation on a type I fibrillar collagen substrate revealed an important role for FXIII and calpain in limiting platelet recruitment into developing aggregates, thereby leading to reduced thrombus formation. These studies define a previously unidentified role for platelet FXIII and calpain in regulating integrin alpha(IIb)beta3 adhesive function. Moreover, they demonstrate the existence of an autoregulatory feedback mechanism that serves to limit excessive platelet accumulation on highly reactive thrombogenic surfaces.

Selective regulation of class I and class II histone deacetylases expression by inhibitors of histone deacetylases in cultured mouse neural cells

The expression of 10 histone deacetylases (HDAC1–10) mRNAs in mouse neuroblastoma Neuro-2a and microglia N9 cell cultures after treatment by inhibitors of HDACs, sodium butyrate and trichostatin A was studied to elucidate whether HDAC inhibitors affect the gene expression of HDACs themselves. Northern blot analysis demonstrated two- to four-fold elevated levels of mRNAs for HDAC1, -3, -5, and -6 after drug treatment in comparison with untreated cells, while mRNA levels for HDAC2 and -7 did not change significantly. In both Neuro-2a and N9 cells the highest increase was observed for HDAC5 and -6 mRNA, whereas, HDAC4 had a prominent increase in mRNA levels after drug treatment only in N9 microglia cell line but not in Neuro-2a. Immunocytochemical examination confirmed that changes in protein levels of HDACs were similar to changes in their mRNA levels. There exists an auto-regulatory feedback loop to the expression levels of several HDACs after inhibition of their biochemical activity, adding several HDAC genes to the list of genes regulated by HDAC inhibitors.

Identification of an Autoregulatory Feedback Pathway Involving Interleukin-1α in Induction of Constitutive NF-κB Activation in Pancreatic Cancer Cells

We previously reported that NF-kappaB is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IkappaBalphaM-mediated inhibition of constitutive NF-kappaB activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-kappaB activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-kappaB is activated in pancreatic cancer remains to be elucidated. In this study, we found that an autocrine mechanism accounts for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1alpha was the primary cytokine secreted by these cells that activates NF-kappaB. Neutralization of interleukin-1alpha activity suppressed the constitutive activation of NF-kappaB and the expression of its downstream target gene, urokinase-type plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1alpha expression is primarily dependent on AP-1 activity, which is in part induced by signaling pathways that are epidermal growth factor receptor-dependent and -independent. In conclusion, our findings suggest a possible mechanism for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.

A link between p73 transcriptional activity and p73 degradation.

The p53 family of proteins includes three members, p53, p63, and p73. The levels and stability of p53 are controlled in large part by MDM2, which can bind the p53 N-terminus and promote its degradation. Because the MDM2 gene is transcriptionally activated by p53, it forms part of an autoregulatory feedback loop that directly links the transcriptional activity of p53 with its degradation. In contrast, little is known about the mechanisms that control p63 or p73 stability. In the current study, p73 deletion or point mutants that lacked transactivation activity were stable compared to wild-type p73. A naturally occurring p73 variant (DeltaNp73) was also stable compared to wild-type p73. Finally, fusion of the VP16-transactivation domain to an inactive, stable p73 mutant restored transactivation function and rendered the mutant protein unstable. These results demonstrate that p73 transactivation activity is necessary for rapid p73 turnover, and suggest that one or more transcriptional targets of p73 may promote its degradation.

CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis.

The circadian clock controls numerous physiological and molecular processes in organisms ranging from fungi to human. In plants, these processes include leaf movement, stomata opening, and expression of a large number of genes. At the core of the circadian clock, the central oscillator consists of a negative autoregulatory feedback loop that is coordinated with the daily environmental changes, and that generates the circadian rhythms of the overt processes. Phosphorylation of some of the central oscillator proteins is necessary for the generation of normal circadian rhythms of Drosophila, humans, and Neurospora, where CK1 and CK2 are emerging as the main protein kinases involved in the phosphorylation of PER and FRQ. We have previously shown that in Arabidopsis, the protein kinase CK2 can phosphorylate the clock-associated protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) in vitro. The overexpression of one of its regulatory subunits, CKB3, affects the regulation of circadian rhythms. Whether the effects of CK2 on the clock were due to its phosphorylation of a clock component had yet to be proven. By examining the effects of constitutively expressing a mutant form of the Arabidopsis clock protein CCA1 that cannot be phosphorylated by CK2, we demonstrate here that CCA1 phosphorylation by CK2 is important for the normal functioning of the central oscillator.

The Bird Clock: A Complex, Multi-Oscillatory and Highly Diversified System

A clock is conceptualized as comprising an input-pacemaker-output system. An overtly measured output, such as the rhythm of activity, feeding or secretion of melatonin, is the consequence of underlying activity of genes at all three levels (input, pacemaker and output) of the clock system. Molecular biochemical studies suggest that the rhythmicity is generated intrinsically within specialized tissues (pacemaker cells) by autoregulatory feedback loops involving gene transcription and post-translational modifications of the ‘clock proteins’. Each tissue that can generate oscillations and produce a stable rhythmic output in synchronization with external (environmental) signals is called a clock, and birds appear to have multiple clocks. Independent input-pacemaker-output systems are present at a minimum of three levels — the retina of the eyes, the pineal gland, and the hypothalamus. This is different from mammals in which input, pacemaker and output are localized in different structures. Further, in birds, each clock interacts with one or more other clocks, and, together, these clocks form a centralized clocking system (CCS). The degree of interdependence between the components of the CCS can vary greatly between species as well as between seasons within an individual. All these properties make the avian CCS a highly complex and diverse timing system. In this article, we will first summarize evidence for the possession of clocks at the three levels (the retina of the eye, the pineal gland, and the hypothalamus), and then present examples that show the complexity and diversity of the avian CCS. We will also analyze how the avian CCS is integrated to produce stable and yet highly adaptive short- and long-term periodic functions.

Math1 controls cerebellar granule cell differentiation by regulating multiple components of the Notch signaling pathway.

Cerebellar granule cells (CGC) are the most abundant neurons in the mammalian brain, and an important tool for unraveling molecular mechanisms underlying neurogenesis. Math1 is a bHLH transcription activator that is essential for the genesis of CGC. To delineate the effects of Math1 on CGC differentiation, we generated and studied primary cultures of CGC progenitors from Math1/lacZ knockout mice. Rhombic lip precursors appeared properly positioned, expressed CGC-specific markers, and maintained Math1 promoter activity in vivo and in vitro, suggesting that Math1 is not essential for the initial stages of specification or survival of CGC. Moreover, the continuous activity of Math1 promoter in the absence of MATH1, indicated that MATH1 was not necessary for the activation of its own expression. After 6, but not 3, days in culture, Math1 promoter activity was downregulated in control cultures, but not in cells from Math1 null mice, thus implying that Math1 participates in a negative regulatory feedback loop that is dependent on increased levels of MATH1 generated through the positive autoregulatory feedback loop. In addition, Math1 null CGC did not differentiate properly in culture, and were unable to extend processes. All Notch signaling pathway receptors and ligands tested were expressed in the rhombic lip at embryonic date 14, with highest levels of Notch2 and Jag1. However, Math1-null rhombic lip cells presented conspicuous downregulation of Notch4 and Dll1. Moreover, of the two transcriptional repressors known to antagonize Math1, Hes5 (but not Hes1) was downregulated in Math1-null rhombic lip tissue and primary cultures, and was shown to bind MATH1, thus revealing a negative regulatory feedback loop. Taken together, our data demonstrate that CGC differentiation, but not specification, depends on Math1, which acts by regulating the level of multiple components of the Notch signaling pathway.

Daily Expression Patterns of Cycle and Clock Genes in the Head of the Silkworm, Bombyx Mori

ABSTRACTClock and cycle (Bmall) genes encode the basic helix-loop-helix-PAS (Period-Aryl-hydrocarbon receptor-Singleminded) proteins that function as transcription factors regulating the transcription/translation-based autoregulatory feedback in the circadian clock of many organisms. In the present study we examined the temporal pattern in the expression of mRNA of Clock (BmClk) and Cycle (BmCyc) genes in the head of Bombyx mori by Northern blot analysis. BmClk gene was co-expressed in the same tissues as those where BmCyc was expressed. In a daily light-dark cycle BmCyc did not show significant difference in expression. Surprisingly unlike the case of Drosophila melanogaster, the levels of BmClk transcript exhibited no prominent circadian oscillation in the moth head. This casts a doubt that the structure and regulatory mechanism of circadian clock may not be uniform among organisms.

EXPOSURE OF MAMMALIAN CELLS TO DIOL EPOXIDES FROM BENZO[A]PYRENE AND DIBENZO[A,L]PYRENE AND EFFECTS ON Mdm2 AND P53

Dibenzo[a,l]pyrene (DBP) is considerably more carcinogenic than benzo[a]pyrene (BP). This fact is most probably related to differences in DNA binding efficiency of their ultimate carcinogenic diol epoxide (DE) intermediates, (+)-anti-BPDE in the case of BP and (−)-anti-DBPDE with DBP; differences in structural features of the DNA adducts and differences in DNA adduct recognition; and the subsequent lesion removal by nucleotide excision repair (NER). The complex signaling network involved in adduct recognition, cell cycle check points, and adduct removal is rapidly emerging. In this study we have focused on the effects of (+)-anti-BPDE and (±)-anti-DBPDE on Mdm2, a protein that regulates p53 stabilization/accumulation in an autoregulatory negative feedback loop. The data available so far indicate that both DEs induce accumulation of Mdm2 in HepG2 cells in concentrations without detectable effects on p53 stabilization. However, BPDE was found to be several orders of magnitude more powerful as inducer than the more complex DBPDE. BPDE caused Mdm2 accumulation at low pM concentrations whereas nM concentrations of DBPDE were required for a similar effect. The Mdm2 accumulation induced by each DE was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. This suggests that phosphorylation of Mdm2 is an essential step in accumulation of the protein. Furthermore, available RT-PCR results indicate that the DEs at higher concentrations (1 μM BPDE and 0.1 μM DBPDE) actually lowered the levels of Mdm2 mRNA indicating transcriptional downregulation of Mdm2. As expected, the effect was associated with p53 phosphorylation on serine 15 and, accordingly, stabilization of the protein.

MDM2 and Prognosis

Abstract The cellular stress response pathway regulated by the p53 tumor suppressor is critical to the maintenance of genomic integrity and to the prevention of oncogenic transformation. Intracellular levels of p53 are tightly regulated by an autoregulatory feedback loop comprised of p53 and MDM2. It might be predicted that disruption of this loop, either through p53 mutation or overexpression of MDM2, would be a negative prognostic marker for cancer development, likelihood of relapse, or response to therapy. In fact, although MDM2 overexpression is common in cancer, it can be both a positive and a negative predictor of outcome in different tumors, and its significance as a biomarker remains controversial. Data from a number of different tumor types are reviewed for the predictive significance of MDM2 expression, along with evidence for different mechanisms of MDM2 overexpression in these different tumors. In light of the biological complexities underlying the p53-MDM2 loop, it is, perhaps, not surprising that no simple paradigm exists that is generally applicable. Much work remains to be done to elucidate the basic mechanisms underlying the physical interactions between the two proteins, the role of protein modifications in altering those interactions, and also the genetic and transcriptional deregulations by which protein levels are altered in human cancers. Only in this way will truly biologically relevant predictive factors emerge.

Regulation of Tie2 Expression by Angiopoietin—Potential Feedback System

To study a potential feedback system in the angiopoietin (Ang)-Tie2 system, the authors examined effects of Ang1 and Ang2 on Tie2 expression on human umbilical vein endothelial cells (HUVECs) with or without stimulation by a potent inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Ang1, but not Ang2, down-regulated Tie2 expression on HUVECs without TNF-alpha stimulation. Both Ang1 and Ang2 attenuated TNF-alpha-induced Tie2 up-regulation. Regulation of Tie2 expression by Ang1 or Ang2 was not dependent on phosphatidylinositol 3-kinase. The Ang-Tie2 system appears to have an autoregulatory feedback system that may be regulating the overall activity of the Tie2 system in both physiological and pathological conditions.