生物谷報道：生物谷在以往的評論中多次提到,，信號轉(zhuǎn)導(dǎo)并非是傳統(tǒng)想象中的一維的,，直線的，單一的模式,，而且非常復(fù)雜的，不僅是直線的,，而且是曲線的,，網(wǎng)絡(luò)的，甚至混沌的,，同時還包括很強的定位和定量特性,。所謂曲線，因為機體內(nèi)蛋白質(zhì)相互作用十分復(fù)雜,，一個信號不可能是單一傳導(dǎo),，而且有許多其它蛋白質(zhì)或信號去增強它，抑制它,，構(gòu)成了一個信號反饋網(wǎng)絡(luò),，從而保證了信號傳導(dǎo)的精確性。如果將信號比作為一個大型機器,，它在每一級傳導(dǎo)過程中,，都會有相應(yīng)的檢測機制，通過正負(fù)反饋的調(diào)節(jié)使信號定量和定位地傳導(dǎo)下去,。本期Science上發(fā)表文章認(rèn)為NF-KB信號由于正負(fù)反饋的調(diào)控,，存在明顯的震蕩現(xiàn)象。生物谷專家認(rèn)為,，信號轉(zhuǎn)導(dǎo)的震蕩是一種普遍現(xiàn)象,，正負(fù)反饋地調(diào)節(jié)決定了震蕩存在的必然性，而且這種震蕩還不是我們想象中這樣簡單,，信號傳導(dǎo)的每一級都會形成一個小的反饋環(huán),，而整個信號又會形成一個大的反饋環(huán),，通過不斷地調(diào)控和震蕩，使信號精確傳導(dǎo)下去,?？梢韵嘈牛谖磥韼啄陜?nèi),，會出現(xiàn)一個理想的數(shù)學(xué)模型研究信號傳導(dǎo)的機理,，它將極大推動人類對信號轉(zhuǎn)導(dǎo)的認(rèn)識。早在2001年生物谷便提出了信號轉(zhuǎn)導(dǎo)研究的定量和定位假說,，在國內(nèi)引起一定的反響,。

另外，信號轉(zhuǎn)導(dǎo)中還存在一個定位傳導(dǎo)問題,。細(xì)胞接受外界信號,，細(xì)胞內(nèi)蛋白質(zhì)傳導(dǎo)這一信號，但信號并非遍布整個細(xì)胞,，而是局限于細(xì)胞的局部,。而且同一信號在細(xì)胞的不同部位，最終產(chǎn)生的效應(yīng)也將是不同的,，這種信號轉(zhuǎn)導(dǎo)的定位特征,，使信號轉(zhuǎn)導(dǎo)變得更為復(fù)雜而有趣。目前有關(guān)信號的定位研究還僅僅局限于神經(jīng)細(xì)胞和心肌細(xì)胞的信號研究,，但相信這種現(xiàn)象同樣存在于所有的細(xì)胞類型中,。

同期Science還有另外一篇報道認(rèn)為細(xì)胞內(nèi)存一個調(diào)控Ca2+/CaM信號的網(wǎng)絡(luò)，其實這一初步研究也提示,，信號轉(zhuǎn)導(dǎo)本身也是受其它信號控制的,，而且是一個復(fù)雜的網(wǎng)絡(luò)信號的調(diào)控。具體見下面的相關(guān)報道,。

NF-B is a family of dimeric transcription factors (usually RelA/p65:p50) that regulates cell division, apoptosis, and inflammation (1). NF-B dimers are sequestered in the cytoplasm of unstimulated cells by binding to IB proteins. NF-B–activating stimuli activate the inhibitor kappa B kinase (IKK) signalosome that phosphorylates IB [at Ser32 and Ser36 on IB (2)] and NF-B [at Ser536 in RelA (3, 4)]. Phosphorylated IB proteins are then ubiquitinated and degraded by the proteasome, liberating NF-B dimers to translocate to the nucleus and regulate target gene transcription.

IB is a transcriptional target for NF-B (5), creating a negative feedback loop (Fig. 1A) in which its delayed expression gives the system similar characteristics to the circadian clock (6) and to ultradian oscillators such as p53 (7, 8) and the segmentation clock (8, 9). IB contains both nuclear localization and export sequences, enabling its nuclear-cytoplasmic (N-C) shuttling. Newly synthesized free IB binds to nuclear NF-B, leading to export of the complex to the cytoplasm (10). This complex, but not free IB, is the target for IB phosphorylation by IKK (11, 12).

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 Fig. 1. Oscillations in NF-B localization. (A) Schematic diagram illustrating the potential mechanism for repeated oscillations in NF-B (p65/RelA) N-C localization. (B) Time-lapse confocal images of SK-N-AS cells expressing RelA-DsRed (red) and IB-EGFP (green) showing single-cell asynchronous N:C oscillations in RelA-DsRed localization after stimulation with 10 ng/ml TNF. The arrow marks one oscillating cell. Times, min; scale bar, 50 µm. [View Larger Version of this Image (78K GIF file)] 

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Oscillations in the temporal response of NF-B activity have been observed by electromobility shift assay (EMSA) only in studies of IBß and  knockout mouse embryonic fibroblast cell populations and have been simulated in a computational model (13). In the absence of time-lapse single-cell analysis, it has remained unclear whether asynchronous single-cell oscillations occur in single cells following NF-B stimulation (8, 14). Like calcium signaling (15), NF-B could be a complex dynamic oscillator using period and/or amplitude to regulate transcription of target genes.

We have used fluorescence imaging of NF-B (RelA) and IB fluorescent fusion proteins (11, 16) to study oscillations in RelA N-C localization (N-C oscillations) in HeLa (human cervical carcinoma) cells and SK-N-AS cells [human S-type neuroblastoma cells that have been associated with deregulated NF-B signaling (17)]. In SK-N-AS cells expressing RelA fused at the C terminus to the red fluorescent protein DsRed (RelA-DsRed) and IB fused at the C terminus to the enhanced green fluorescent protein EGFP (IB-EGFP) (Fig. 1B and Fig. 2A), 96% showed an NF-B nuclear translocation response to tumor necrosis factor alpha (TNF) stimulation and 72% showed long-term N-C oscillations in RelA-DsRed localization. Oscillations with a typical period of 100 min continued for >20 hours after continuous TNF stimulation, damping slowly. In transfected cells expressing RelA-DsRed and control EGFP (Fig. 2C), 97% responded and 91% of cells showed N-C oscillations. These oscillations appeared more synchronous between cells in the first three cycles compared with cells that also expressed IB-EGFP, which suggests that the system was sensitive to variation in IB levels, thus contributing to the degree of cell-to-cell asynchrony. When HeLa cells were continually stimulated with TNF (Fig. 2D), 86% of the cells responded and 30% exhibited up to three detectable N-C oscillations that were markedly damped. However, when TNF was added to SK-N-AS cells (Fig. 2B) or HeLa cells (Fig. 2E) as a 5-min pulse, a single peak of nuclear occupancy was observed with no subsequent cycles of RelA movement.

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 Fig. 2. Analysis of the dynamics of NF-B localization and B-dependent reporter gene expression. (A to F) Time course of N:C localization of RelA-DsRed in cells co-expressing IB-EGFP [(A), (B), (D), (E), and (F)] or EGFP control (C). N:C ratioinRelA-DsRed fluorescence was normalized to highest peak intensity. The peak N:C ratio was expressed as the average value for each set of four cells. Data from each cell is represented by a different colored line. (G to K) Luminescence imaging (RLU, relative light units) of the dynamics of B-dependent luciferase reporter activity represented as a different colored line for each of four different cells. The black line represents the average of the cells. [(A), (C), and (G)] SK-N-AS cells treated with continual 10 ng/ml TNF. [(D) and (I)] HeLa cells treated with continual 10 ng/ml TNF. [(B) and (H)] SK-N-AS cells treated with a 5-min TNF pulse. [(E) and (J)] HeLa cells treated with a 5-min TNF pulse. [(F) and (K)] SK-N-AS cells treated with 20 µM of etoposide. The black bar above each graph is a representation of the duration of TNF treatment. For images of data in [(B) to (F)], (G), and (I), see figs. S6 to S11. [View Larger Version of this Image (48K GIF file)] 

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TNF treatment induced endogenous RelA localization patterns in cells, consistent with increasingly asynchronous N-C oscillations (fig. S3). Western blot analysis (figs. S4 and S5) showed that SK-N-AS and HeLa cells continually treated with TNF gave biphasic dynamics of total IB, phosphorylated IB (Ser32 phospho-IB), and phosphorylated RelA (Ser536 phospho-RelA). In HeLa cells, phosphoprotein expression levels diminished more rapidly than in the SK-N-AS cells (fig. S5). A 5-min TNF pulse directed transient accumulation of Ser32 phospho-IB and Ser536 phospho-RelA (fig. S4B). These data support the hypothesis that loss of IKK activity (due to TNF removal) results in loss of N-C oscillations and that dephosphorylation of RelA occurs rapidly without persistent IKK activity. When SK-N-AS cells were treated with an alternative stimulus, the topoisomerase II inhibitor etoposide (VP16), 37% of the cells responded and 24% showed N-C oscillations. Etoposide-induced N-C oscillations had lower amplitude than those induced by TNF, peaking after 300 min and then diminishing (Fig. 2F). The IB and RelA phosphoprotein expression levels after etoposide treatment (fig. S4C) corresponded to the timing of N-C oscillations.

We investigated whether N-C oscillation persistence influenced the dynamics of NF-B–regulated gene expression using real-time imaging of firefly luciferase activity (18) driven by a B (5 x consensus site) promoter. SK-N-AS cells exhibited stable luminescence for more than 25 hours in the continual presence of TNF (Fig. 2G). HeLa cells showed a transient peak 10 hours after TNF treatment that decayed by 20 hours (Fig. 2I). In SK-N-AS (Fig. 2H) or HeLa cells (Fig. 2J) treated with a 5-min TNF pulse, a more transient peak of luminescence occurred after 5 hours, which decayed by 10 hours. Etoposide treatment of SK-N-AS cells elicited a lower luminescence signal, reaching a peak at 15 hours after treatment (Fig. 2K). With each stimulus, the kinetics of NF-B oscillations and maintenance of phosphoprotein levels appeared closely related to the kinetics of gene expression. Thus, persistent NF-B oscillations appear to maintain NF-B–dependent gene expression.

Analysis of successive peaks of RelA nuclear occupancy (figs. S13 and S14 and Fig. 3, E and F) showed that N-C oscillation damping and successive peak timing were highly reproducible, but because of phase differences, this was not apparent at the population level. However, the pattern of peak timing and amplitudes was different between HeLa and SK-N-AS cells. The expression of IB–EGFP affected the amplitude and peak timing of the N-C oscillations (fig. S14). To study the role of IB synthesis rate on N-C oscillations, the rate of NF-B–regulated IB transcription was modulated. IB-EGFP expression was driven by the B (5 x consensus site) promoter and expressed in HeLa cells together with a fusion protein between RelA and the modified red fluorescent protein DsRed-Express (RelA-DsRed-Express). Continual TNF stimulation elicited oscillations in IB-EGFP expression out of phase with the RelA N-C oscillations (Fig. 3, A and B). This caused a statistically significant delay in the timing of nuclear RelA peaks 1, 2, and 3 (Fig. 3F). The amplitude was also slightly reduced for peaks 2 and 3 in the presence of the B-IB-EGFP expression vector (Fig. 3E).

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 Fig. 3. NF-B–directed oscillations in IB expression. Experimental and computational analysis of factors affecting the amplitude and period of oscillations. (A, B, E, and F) HeLa cells were transfected to express RelA-DsRed-Express and IB-EGFP under the control of either the consensus B promoter or a control B promoter vector. Cells were stimulated with continual 10 ng/ml TNF. (A) Confocal time course of one typical cell showing oscillations in both RelA-DsRed-Express (red) localization and IB-EGFP (green) expression. Scale bar, 50 µm. (B) Analysis of three typical cells showing RelA-DsRed-Express N:C ratio and cytoplasmic and nuclear IB-EGFP levels. (C) The simulated time-dependent nuclear localization of NF-B for successively increasing the NF-B–regulated IB transcription rate constant by two orders of magnitude on either side of the standard rate constant (reaction 28 in the computational model, table S1) is shown by 41 lines changing in regularly increasing log intervals from blue to green to yellow to red (scanned after equilibration). (D) The peak amplitudes (A1 to A6) and timings (T1 to T6) of the first six simulated peaks for different rate constant values for NF-B regulated IB transcription [as determined from data in (C)]. (E) Experimentally determined relative amplitude (N:C ratio) of successive RelA-DsRed-Express oscillations in HeLa cells continually stimulated with TNF. Peak 1 set to 100%; subsequent peaks show relative amplitude ±SEM). (F) Average timing between successive peaks (±SEM) of successive N-C oscillations in RelA-DsRed-Express. (G) Simulated peak timings for 1x, 2x, 5x, and 10x standard reaction rate constant for NF-B–regulated IB transcription (reaction 28 in computational model, table S1). The parameter was changed before the equilibration period. [View Larger Version of this Image (68K GIF file)] 

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To investigate parameters affecting the oscillation dynamics, we used a computational model (13) that predicted NF-B oscillations with a similar period and damping as those observed here. From this model, we noted that changes in just two molecular species (variables), free IKK and IB, were intimately coupled to the oscillation dynamics of nuclear NF-B (fig. S16). Transfection with the B-IB-EGFP expression vector (Fig. 3, A, B, E, and F) was equivalent to increasing the rate of NF-B–dependent IB transcription; thus, we chose to study the effect of this parameter in the model (reaction 28 in table S1; Fig. 3, C and D; and fig. S17). See (19) for analysis of some other related parameters (figs. S18 and S19). As the rate of this reaction was increased, there was a delay in simulated peaks 2 and onward (Fig. 3, C, D, and G). Thus, the computational analysis showed the effects of this reaction rate to be similar to those seen in the experimental studies. One discrepancy between the computational model and the experimental data was the unpredicted delay in experimentally observed peak 1 caused by B-IB-EGFP transfection (Fig. 3, F and G). It is unclear how the two cell types studied differ with respect to the values of the parameters used in the model. Given that the oscillations are naturally asynchronous between cells and that this might be associated with varying levels of IB proteins (13) or a lack of optimization of the preequilibration step in the model, this may explain why the timing of peak 1 was imperfectly predicted.

The amplitude of oscillations in IB-EGFP when expressed under the control of the B promoter was not directly related to the amplitude of the preceding peak in RelA nuclear localization. In many HeLa cells, peak 2 or 3 in RelA localization was small in amplitude (Fig. 3B) compared with peak 1 (and would not have been observed in asynchronous populations). Nevertheless, these oscillations led to easily observable IB-EGFP responses. Thus, persistence of NF-B oscillations maintains NF-B–dependent transcription. However, NF-B translocation cannot be the only factor regulating transcriptional activation (a property of the whole system), and further NF-B activating and inactivating reactions, including modifications of RelA by phosphorylation (3, 4, 20), acetylation (21), or prolyl isomerization/targeted degradation (22), have also been described. The cessation of NF-B–dependent transcription in the nucleus, independent of nuclear export (11), might occur as a consequence of RelA inactivation. Thus, NF-B oscillations could repeatedly deliver newly activated NF-B into the nucleus, maintaining a high nuclear ratio of active:inactive NF-B. To investigate this hypothesis, we used the CRM1-dependent nuclear export inhibitor leptomycin B (LMB) to trap RelA in the nucleus of SK-N-AS cells (Fig. 4, A and B). This resulted in transitory B-dependent luciferase reporter gene expression (11) that peaked after 5 hours (Fig. 4C). Western blot analysis indicated a transient increase in Ser32 phospho-IB expression after 5 min, with no subsequent recovery (Fig. 4E). Ser536 phospho-RelA expression was maximal at 5 min after stimulation and decayed to the threshold of detection by 180 min (in contrast to cells treated with constant TNF, Fig. 4D). These data support the hypothesis (23) that rapid dephosphorylation of NF-B in the nucleus [by PP2A activity (24)] may be a key factor in the switch-off of NF-B–dependent gene expression.

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 Fig. 4. Effect of nuclear export inhibition on the dynamics of RelA localization, B-dependent reporter gene expression, and NF-B phosphoprotein expression. SK-N-AS cells were treated with continuous 10 ng/ml TNF and 10 ng/ml LMB (unless stated). (A) Time-lapse confocal images of RelA-EGFP localization. (B) Time course of RelA-EGFP localization expressed as N:C fluorescence ratio (each colored line represents data from one of four single cells). (C) B-dependent luciferase reporter gene expression (each colored line represents data from one of four single cells, and the black line represents the average). (D) Western blot analysis of Ser32 phospho-IB (P-IB), total IB (IB), Ser536 phospho-RelA (P-RelA), and total RelA (RelA) protein levels in SK-N-AS cells stimulated with continual 10 ng/ml TNF for the indicated times before analysis. (E) Western blot analysis of SK-N-AS cells stimulated with continual 10 ng/ml TNF and 18 nM LMB for the indicated times before analysis. 

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We propose that oscillations in NF-B localization coupled to cycles of RelA and IB phosphorylation maintain NF-B–dependent gene expression. Calcium spikes at intervals as long as 30 min have been shown to maintain NF-B activity in T cells (25). The decoding of this [Ca2+] spike frequency might be related to the observed kinetics of oscillatory transcription factor shuttling and regulation (26). Specific, nonlinear "network motifs" can decode frequencies rather than amplitudes (27). Therefore, the signal-processing elements of the NF-B signaling pathway, and its interaction with other dynamic signaling systems, may involve the encoding and decoding of specific time-varying signals. Such temporal encoding could avoid undesirable cross talk between cellular signaling pathways that share common components. Furthermore, oscillatory phosphorylation of RelA at Ser536 appears to be a consequence of its shuttling between the cytoplasm and the nucleus. Oscillatory modifications at other regulatory amino acids in RelA (21, 28) might also occur as a consequence of N-C oscillations, whereas changes in N-C oscillation frequency and persistence might explain differential regulation of cell fate in response to different stimuli. Thus, in common, and perhaps in combination, with other oscillatory transcription factor pathways such as p53 (7, 8), NF-B may constitute a complex analog-to-digital coding system that regulates cell fate.

文章來源：Oscillations in NF-B Signaling Control the Dynamics of Gene Expression
D. E. Nelson, A. E. C. Ihekwaba, M. Elliott, J. R. Johnson, C. A. Gibney, B. E. Foreman, G. Nelson, V. See, C. A. Horton, D. G. Spiller, S. W. Edwards, H. P. McDowell, J. F. Unitt, E. Sullivan, R. Grimley, N. Benson, D. Broomhead, D. B. Kell, and M. R. H. White
Science 22 October 2004: 704-708.

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