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Core promoters: active contributors to combinatorial gene regulation.

In metazoans, thousands of protein-coding genes must be differentially expressed in specific cell types, during development, and in response to a wide variety of extracellular signals. Combinatorial gene regulation strategies are required to generate these diverse expression patterns because only a limited number of transcription factors can be encoded by a limited genome. For a gene to be activated, transcription factors must bind distant control regions and promote the decondensation of repressed chromatin. Then, factors bound to distant control regions and the promoter must stimulate the remodeling of individual nucleosomes and transcription initiation by RNA polymerase II, via effective communication with nucleosome remodeling complexes, coactivator complexes, and the general transcription machinery (Lemon and Tjian 2000). Another important feature of combinatorial regulation is the requirement for several distinct transcription factors to activate a gene (Merika and Thanos 2001). By employing combinations of factors, the number of gene expression patterns that can be achieved is greatly enhanced. Although combinatorial regulation has been widely studied, one potential contributor has received relatively little attention: the core promoter, which is located between approximately −35 and +35 relative to the transcription start of a metazoan gene. One reason the core promoter generally was not considered to be an active contributor to combinatorial regulation is historical; when the first protein-coding genes were isolated, virtually every gene, regardless of its expression pattern, contained an A/T-rich sequence 25–30 base pairs (bp) upstream of the transcription start site (Breathnach and Chambon 1981). This sequence, with the consensus TATAAA, was called the TATA box. Following the development of functional assays, mutations in TATA boxes were found to reduce transcription initiation and prevent the proper positioning of transcription start sites. Based on these early observations, it was expected that a similar core promoter structure would be found in every cellular gene. The regulation of transcription was expected to rely exclusively on DNA-binding proteins that interact with distal promoters and enhancers. Today, this simplistic picture of the structure of core promoters for protein-coding genes has been replaced by a level of complexity that is not yet fully understood or appreciated. Most likely, the initial similarity resulted from the fact that the first promoters for RNA polymerase II were identified in DNA viruses and highly expressed cellular genes, which often contain TATA boxes. As more and more promoters for cellular genes have been isolated, the extensive similarity has vanished. In Drosophila, most core promoters for protein-coding genes fall into two distinct classes (Burke et al. 1998; Kutach and Kadonaga 2000). Roughly half contain a TATA box 25 to 30 bp upstream of the transcription start site combined with an initiator (Inr) element overlapping the start site. The other half contain an Inr element combined with a downstream promoter element (DPE), which is located approximately 30 bp downstream of the start site. Importantly, all three of these elements serve as recognition sites for subunits of transcription factor IID (TFIID), which contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs) (for review, see Burke et al. 1998; Smale et al. 1998). In mammals, core promoter structure appears to be even more diverse. Precise calculations have been difficult because transcription start sites have been determined accurately for only a small fraction of genes. Nevertheless, the available data suggest that (1) a smaller percentage of mammalian promoters than Drosophila promoters contain TATA boxes, (2) TATA boxes are paired with Inr elements in a smaller percentage of mammalian promoters, (3) DPE elements exist in mammalian promoters, but have been difficult to identify, and (4) many promoters, including a number of promoters within CpG islands, appear to lack all three of these core elements. The diversity of core promoter structure leads to two general questions that are of fundamental importance for an understanding of transcriptional control. First, what are the similarities and differences between the mechanisms of transcription initiation catalyzed by the various core promoter classes? Second, why does core promoter diversity exist? Although the first question has been explored in a number of studies (for reviews, see Burke et al. 1998; Smale et al. 1998; Lemon and Tjian 2000), little is known about the second. One possible reason for core promoter diversity is that the different classes of core promoters may have evolved E-MAIL steves@hhmi.ucla.edu; FAX (310) 206-8623. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.937701.

In metazoans, thousands of protein-coding genes must be differentially expressed in specific cell types, during development, and in response to a wide variety of extracellular signals. Combinatorial gene regulation strategies are required to generate these diverse expression patterns because only a limited number of transcription factors can be encoded by a limited genome. For a gene to be activated, transcription factors must bind distant control regions and promote the decondensation of repressed chromatin. Then, factors bound to distant control regions and the promoter must stimulate the remodeling of individual nucleosomes and transcription initiation by RNA polymerase II, via effective communication with nucleosome remodeling complexes, coactivator complexes, and the general transcription machinery (Lemon and Tjian 2000). Another important feature of combinatorial regulation is the requirement for several distinct transcription factors to activate a gene (Merika and Thanos 2001). By employing combinations of factors, the number of gene expression patterns that can be achieved is greatly enhanced.
Although combinatorial regulation has been widely studied, one potential contributor has received relatively little attention: the core promoter, which is located between approximately −35 and +35 relative to the transcription start of a metazoan gene. One reason the core promoter generally was not considered to be an active contributor to combinatorial regulation is historical; when the first protein-coding genes were isolated, virtually every gene, regardless of its expression pattern, contained an A/T-rich sequence 25-30 base pairs (bp) upstream of the transcription start site (Breathnach and Chambon 1981). This sequence, with the consensus TATAAA, was called the TATA box. Following the development of functional assays, mutations in TATA boxes were found to reduce transcription initiation and prevent the proper positioning of transcription start sites. Based on these early observations, it was expected that a similar core promoter structure would be found in every cellular gene. The regulation of transcription was expected to rely exclusively on DNA-binding proteins that interact with distal promoters and enhancers.
Today, this simplistic picture of the structure of core promoters for protein-coding genes has been replaced by a level of complexity that is not yet fully understood or appreciated. Most likely, the initial similarity resulted from the fact that the first promoters for RNA polymerase II were identified in DNA viruses and highly expressed cellular genes, which often contain TATA boxes. As more and more promoters for cellular genes have been isolated, the extensive similarity has vanished.
In Drosophila, most core promoters for protein-coding genes fall into two distinct classes (Burke et al. 1998;Kutach and Kadonaga 2000). Roughly half contain a TATA box 25 to 30 bp upstream of the transcription start site combined with an initiator (Inr) element overlapping the start site. The other half contain an Inr element combined with a downstream promoter element (DPE), which is located approximately 30 bp downstream of the start site. Importantly, all three of these elements serve as recognition sites for subunits of transcription factor IID (TFIID), which contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs) (for review, see Burke et al. 1998;Smale et al. 1998). In mammals, core promoter structure appears to be even more diverse. Precise calculations have been difficult because transcription start sites have been determined accurately for only a small fraction of genes. Nevertheless, the available data suggest that (1) a smaller percentage of mammalian promoters than Drosophila promoters contain TATA boxes, (2) TATA boxes are paired with Inr elements in a smaller percentage of mammalian promoters, (3) DPE elements exist in mammalian promoters, but have been difficult to identify, and (4) many promoters, including a number of promoters within CpG islands, appear to lack all three of these core elements.
The diversity of core promoter structure leads to two general questions that are of fundamental importance for an understanding of transcriptional control. First, what are the similarities and differences between the mechanisms of transcription initiation catalyzed by the various core promoter classes? Second, why does core promoter diversity exist? Although the first question has been explored in a number of studies (for reviews, see Burke et al. 1998;Smale et al. 1998;Lemon and Tjian 2000), little is known about the second.
One possible reason for core promoter diversity is that the different classes of core promoters may have evolved as functionally equivalent landing pads for the general transcription machinery. That is, during the evolution of a complex genome, an interaction site for at least one component of the general machinery may have been required in each promoter, but the recognition site(s) that evolved in a given promoter may have had no special significance other than to facilitate the binding and proper positioning of the general machinery. Early support for this hypothesis was provided by the observation that core promoter elements are functionally interchangeable in the context of simple, synthetic promoters analyzed in cell-free transcription and transient transfection assays. For example, either a TATA box or an Inr element was sufficient for strong, accurately initiated transcription stimulated by transcription factor Sp1 (O'Shea-Greenfield and . Although TATAdirected transcription initiated 25 bp downstream of the TATA box and Inr-directed transcription initiated from within the Inr itself, the elements were otherwise functionally equivalent. A second hypothesis to explain the existence of diverse core promoters is that they may be recognized by different proteins or protein complexes, each of which contributes to the transcription of a relatively small percentage of genes. Support for this hypothesis emerged from the properties of TBP-related factors (TRFs; Dantonel et al. 1999;Lemon and Tjian 2000) and TFTC (Wieczorek et al. 1998). The TFTC (TBP-free TAFII-containing) complex contains several TAFs in common with TFIID and can substitute for TFIID in cell-free transcription assays, yet TFTC lacks TBP. Although the absence of TBP suggests that TFTC may prefer TATA-less promoters, TFTC and TFIID appear to stimulate transcription with equal efficiency from TATA-containing and TATA-less promoters (Wieczorek et al. 1998). These results suggest that TFTC may not discriminate between core promoters with different structures, although it is likely to make other important contributions to differential gene regulation.
TRF1 recognizes a sequence that differs from the consensus TATA box, perhaps explaining the evolution of a class of promoters containing a non-consensus TATA sequence (Holmes and Tjian 2000). However, most of the diversity within metazoan core promoters appears to involve the variable occurrence of consensus or near-consensus TATA, Inr, and DPE elements. As mentioned above, all three of these consensus elements are recognized by subunits of the ubiquitous TFIID complex. In fact, TFIID complexes purified from cells expressing an epitope-tagged TBP are equally competent for transcription from and/or binding to TATA, TATA-Inr, Inr, and Inr-DPE promoters (Smale et al. 1990;Burke and Kadonaga 1996;Emami et al. 1997). Although these results do not rule out the possibility that the purified preparations contain multiple TBP-containing complexes with different combinations of TAFs or the possibility that alternative complexes are used in a physiological setting, they suggest that the most common core promoter elements serve as recognition sites for the same TFIID complex. Thus, although TRFs contribute to core promoter diversity ( Fig. 1D), they are unlikely to be responsible for the variable occurrence of the most common core promoter elements.
The most intriguing hypothesis to explain the variable occurrence of TATA, Inr, and DPE elements is that the diversity makes an important contribution to combinatorial gene regulation. Selective communication between transcription factors bound to distal sites and the core promoter could be of considerable benefit to combinatorial strategies. The results of Butler and Kadonaga (2001) in this issue provide strong support for this hy-Figure 1. Four strategies by which core promoters may contribute to combinatorial regulation. (A) By possessing intrinsic preferences for specific core promoter classes, some enhancers may influence transcription initiation from only a subset of genes within their stimulatory range (Butler and Kadonaga 2001). (B) As a variation of strategy A, competition between core promoters may restrict the stimulatory effects of some enhancers (Ohtsuki et al. 1998). When stimulation of the preferred TATA-Inr promoter is eliminated by insertion of an insulator (Ohtsuki et al. 1998) or, hypothetically, by specific repression of that promoter, the enhancer gains the capacity to stimulate transcription from the Inr-DPE promoter. (C) By possessing intrinsic preferences for a specific core promoter class, some transcriptional activators may stimulate transcription only when bound to promoters that are representative of that class (Ernst et al. 1996;Garraway et al. 1996). (D) As a variation of strategy C, some transcriptional activators, and possibly some enhancers, may possess intrinsic preferences for core promoters that interact with TRFs (Holmes and Tjian 2000). pothesis. Although a role for core promoter diversity in combinatorial regulation was suggested in previous studies, the current study supports an intriguing new version of the hypothesis and involved an elegant strategy that strengthens the physiological relevance of the results and sets the stage for future advances.

Role of core promoters in restricting the stimulatory capacity of enhancers
The study by Butler and Kadonaga (2001) was designed to examine one possible strategy by which core promoters in Drosophila could contribute to combinatorial regulation. The hypothesis is based on the knowledge that enhancers can stimulate transcription from promoters at great distances. In a complex genome, multiple enhancers may be in "striking distance" of a given promoter and multiple promoters may be within the stimulatory range of a given enhancer. To optimize the potential for com binatorial regulation, a means of limiting enhancer stimulation to a subset of promoters in its vicinity would be of considerable benefit. Selective communication between promoters and enhancers has been observed previously (Li and Noll 1994;Merli et al. 1996), but the mechanisms responsible for selectivity were not established. The hypothesis tested by Butler and Kadonaga (2001) was that core promoter diversity contributes to the selective communication between promoters and enhancers. To test this hypothesis, the objective was to determine whether the stimulatory capacity of an enhancer can indeed be restricted to a specific core promoter class. Because the study was performed in Drosophila, the authors were particularly interested in the differences between the TATA-Inr and Inr-DPE classes (Kutach and Kadonaga 2000). Although the properties of a few known enhancers could have been examined in a basic transfection assay, the authors instead designed a strategy that circumvented important obstacles and greatly enhanced the relevance of the results.
One potential obstacle was that only a small subset of enhancers may exhibit core promoter selectivity. To overcome this obstacle, the authors used an enhancer trap strategy and P-element-mediated transformation to introduce promoter-reporter cassettes into numerous locations within the Drosophila genome. The ability to screen a large series of transgenic Drosophila lines greatly increased the probability of identifying enhancer activities that are selective for a TATA-Inr or Inr-DPE core promoter. The second obstacle was that the results would be interpretable only if the two core promoters were examined in precisely the same genomic location. A waffle vector designed by Siegal and Hartl (1997) was ideal for this purpose. The construct that was prepared contained both TATA-Inr and Inr-DPE core promoters, with each core promoter linked to a green fluorescent protein (GFP) reporter gene. When each transgenic line was crossed with another line expressing the Cre recombinase, recombination at loxP sites within the construct resulted in the excision of the Inr-DPE cassette, leaving the TATA-Inr cassette in the genome. When the same lines were crossed with a line expressing the FLP recom-binase, recombination at FRT sites within the construct excised the TATA-Inr cassette, leaving the Inr-DPE cassette in exactly the same genomic location.
From an analysis of 18 pairs of lines, 3 lines expressed much higher concentrations of GFP mRNA from the Inr-DPE promoter and 1 line expressed much higher concentrations from the TATA-Inr promoter. Equivalent expression from the two promoters was observed in the other lines. An examination of the transcription start sites validated the results. The only reasonable explanation for the different activities was that surrounding control regions, presumably enhancers, preferentially stimulate transcription from one core promoter class. Thus, these results provide strong support for the hypothesis that the two types of core promoters exist, at least in part, for the purpose of limiting enhancer effects to a specific subset of promoters (Fig. 1A).
It may be worthwhile noting one additional implication of the results: They support the notion that distant control regions do not exist solely for the purpose of altering chromatin structure. Rather, the core promoter selectivity can be interpreted as strong evidence that enhancer-bound factors must communicate directly or indirectly with factors bound to the core promoter.

Additional contributions to combinatorial regulation
Although the results of Butler and Kadonaga (2001) represent compelling evidence that core promoter diversity contributes to combinatorial regulation, initial support for this general hypothesis was provided by previous studies. One notable study by Ohtsuki et al. (1998) tested a hypothesis that was similar to that tested by Butler and Kadonaga. The Ohtsuki study was inspired by the observation that the AE1 enhancer of the Drosophila Hox gene cluster is positioned between the fushi tarazu (ftz) and Sex combs reduced (Scr) promoters, but activates transcription only from the ftz promoter. The ftz promoter contains a consensus TATA box and the Scr promoter, a classic Inr-DPE combination. This observation led to the hypothesis that AE1 preferentially stimulates TATA-containing promoters. Analysis of an extensive series of transgenic Drosophila lines confirmed this hypothesis. Interestingly, however, the TATA preference of the AE1 region was dependent on competition between the two classes of promoters. That is, AE1 was perfectly competent for activation of an Inr-DPE promoter when the nearby TATA promoter was compromised (Fig. 1B).
In the study by Butler and Kadonaga (2001), the enhancer activities that were monitored appear to possess intrinsic core promoter preferences that do not require competition. The authors will need to address the possibility that selectivity requires competition with endogenous promoters in the vicinity of the integration site. A requirement for competition seems unlikely, however, because enhancers with preferences for each core promoter class were detected. In contrast, the enhancers that required competition for selectivity consistently preferred a TATA box.
The promoter for the murine terminal transferase

GENES & DEVELOPMENT 2505
Cold Spring Harbor Laboratory Press on July 22, 2018 -Published by genesdev.cshlp.org Downloaded from (TdT) gene was the focus of another study that suggested a different route through which core promoter diversity can contribute to combinatorial regulation. The −30 region of this promoter is G/C-rich and does not contribute to promoter function (Smale and Baltimore 1989;Garraway et al. 1996). Instead, a consensus Inr element is responsible for core promoter activity and for dictating the location of the transcription start site. Because the in vitro activity of this promoter requires the TFIID complex (Martinez et al. 1995), a reasonable expectation was that a TATA box engineered at −30 would be able to substitute for the Inr at the start site. Surprisingly, when a TATA box was inserted at −30 and the Inr was disrupted by a point mutation, the promoter was completely inactive (Garraway et al. 1996). As a control, Sp1 sites were inserted into the promoter in place of the distal TdT promoter sequences. When transcription was driven by Sp1 instead of by the natural activators, promoter activity was stronger in the presence of the TATA box than the Inr. These results revealed that the function of the native TdT promoter requires the presence of an Inr and that this preference is due, at least in part, to an Inr preference of transcription factors bound to the distal promoter. Subsequent studies suggested that Elf-1, a critical Ets-family protein that binds 60 bp upstream of the start site, possesses an intrinsic preference for Inrcontaining promoters (Ernst et al. 1996). A DPE-like element located downstream of the Inr also contributes to the preference .
Because the Inr preference of the TdT promoter was observed in vitro in the absence of an enhancer, this result suggests other means by which core promoters can contribute to combinatorial regulation. The simplest scenario is that core promoter selectivity limits the ability of a transcription factor like Elf-1 to activate transcription through promoters containing its recognition sequence (Fig. 1C). The consensus recognition site for Elf-1 is very similar to that of other members of the Ets family, suggesting that DNA-binding specificity is insufficient to restrict Elf-1's functions to the intended set of target genes. By restricting its transactivation capabilities to promoters containing Inr elements, Elf-1 would be ineffective when bound to promoters containing only a TATA box. Thus, an Ets consensus site within a promoter containing only a TATA box would contribute to activation only when it interacts with a different Etsfamily protein that is competent for TATA-mediated transcription.
In addition to Elf-1, a strong preference for an Inr element has been observed with the glutamine-rich activation domains of Sp1 (Emami et al. 1995). Although interesting from a mechanistic perspective, the biological relevance of this preference is uncertain because fulllength Sp1 is a potent activator of promoters containing either a TATA box or Inr element (Emami et al. 1995). A preference for activation of TATA-containing promoters was described for c-Fos and a specific activation domain was found to be responsible for this preference (Metz et al. 1994); deletion of this domain resulted in comparable activation of promoters containing either a TATA box or Inr element. Transcriptional repression by the p53 protein was also found to depend on the presence of a TATA box within the core promoter (Mack et al. 1993). Synthetic promoters containing TATA boxes were potently repressed by p53, but comparable promoters containing an Inr element instead of a TATA box were resistent to repression.
Finally, some promoters and isolated activators exhibit strong preferences for combinations of core promoter elements, whereas others are equally potent when only one core element is present. For example, strong activation by full-length Sp1 occurs with a core promoter containing either a TATA box, an Inr element, or both TATA and Inr elements, whereas GAL4-VP16 activates transcription much more strongly when both elements are present (Emami et al. 1995). A similar preference for core promoters containing both elements was observed with the bovine papillomavirus E2 transactivator (Ham et al. 1994). The biological benefit of combining two core elements for the purpose of strengthening a promoter is apparent in the Drosophila Adh gene, which is transcribed from two different promoters. The preferential utilization of the distal promoter in early stages of development is due, at least in part, to the presence of both TATA and Inr elements (Hansen and Tjian 1995). By contrast, the weaker proximal promoter appears to contain only a TATA box. Although these final examples differ in some respects from the preference of an enhancer, promoter, or activator for a specific core element, they help to illustrate the various means by which core promoter diversity can contribute to combinatorial regulation.

Mechanistic basis of core promoter selectivity
The biochemical mechanisms responsible for the core promoter preferences of enhancers, promoters, and activators remain largely undefined. Therefore, a discussion of this issue must rely primarily on speculation. As mentioned above, the mechanisms responsible for these preferences are not immediately obvious because the TATA box, Inr, and DPE all appear to be recognized by subunits of the same TFIID complex. The most extensive mechanistic insight is perhaps related to the preference of some activators for two core elements (e.g., TATA-Inr) as opposed to one (TATA or Inr). Biochemical studies have shown that a TFIID-TFIIA complex binds cooperatively to the two elements (Emami et al. 1997). This observation suggests that activators like GAL4-VP16 and E2, which prefer two elements, stimulate transcription via a mechanism that benefits from the enhanced affinity of TFIID-TFIIA for the TATA-Inr combination. These activators may be unable to recruit TFIID-TFIIA to weaker core promoters or may be unable to stabilize TFIID-TFIIA binding after recruitment. In contrast, Sp1 activation derives little benefit from the enhanced affinity of TFIID-TFIIA for the TATA-Inr combination, perhaps because it recruits or stabilizes the TFIID-TFIIA complex more strongly than GAL4-VP16 or E2.
Preferences for a specific core promoter element, such as a TATA box, Inr, or DPE, are more difficult to explain.
It is well established that different TAFs are responsible for selective interactions with different core promoter elements. For example, hTAFII150 and hTAFII250 contribute to the selective recognition of promoters containing Inr elements, and dTAFII60 contributes to the selective recognition of promoters containing DPE elements (Burke and Kadonaga 1997;Chalkley and Verrijzer 1999). In addition, disruption of genes encoding TAFs led to reduced transcription of specific sets of genes, with core promoters responsible for some of the selective effects (for review, see Green 2000). The relevance of these results to the core promoter preferences of enhancers, promoters, and activators is uncertain, however, primarily because the TAFs that have been studied are all components of the same TFIID complex. Unless different classes of core promoters are recognized by different forms of TFIID that carry out selective interactions with transcriptional activators, the TAF results do not provide a clear explanation for the core promoter preferences.
A more attractive hypothesis is that the core promoter preferences involve other differences between the mechanisms of transcription initiation from different core promoter classes. Of particular interest are factors that exhibit unique functions with specific classes of core promoters but that are not considered to be components of the general transcription machinery. The proteins that are most intriguing are TIC-2, TIC-3, and NC2. TIC-2 and TIC-3 were identified as biochemical activities in HeLa cell extracts that were required for transcription from a core promoter containing only an Inr (Martinez et al. 1998). Interestingly, these factors, which have not been cloned or identified, had no effect on the strength of TATA or TATA-Inr core promoters. Although there is no evidence that TIC-2 and TIC-3 contribute to the core promoter preferences of transcriptional activators, their selective involvement in the activity of one core promoter class makes them attractive candidates for this function. It is noteworthy that biochemical studies identified two other factors that are essential for Inr activity, TAFII150 (CIF150) and TIC-1 (Verrijzer et al. 1995;Kaufmann et al. 1996Kaufmann et al. , 1998Martinez et al. 1998). These factors are less likely to be responsible for the Inr preference of an activator because, unlike TIC-2/3, they appear to stimulate transcription from all core promoter classes.
NC2 is perhaps the most intriguing example of a factor that may be involved in core promoter selectivity. Unlike the factors described above, it possesses opposing activities with TATA-Inr and Inr-DPE promoters. It was originally described as an inhibitor of TATA-Inr promoters (for review, see Maldonado et al. 1999) but was recently purified from Drosophila embryo extracts as an essential and selective activator of Inr-DPE promoters (Willy et al. 2000). The opposing functions of NC2 raise the possibility that it could contribute to the core promoter preference of a transcriptional activator; if the activator stimulates incorporation of NC2 into the preinitiation complex, it most likely would exhibit a strong preference for an Inr-DPE promoter.
Although factors like NC2 and TIC-2/3 have the po-tential to contribute to core promoter preferences of transcriptional activators, selectivity factors are not necessarily required for these preferences. Instead, activators may influence parameters of the transcription initiation reaction that are important for only one core promoter class. For example, the rate-limiting steps for initiation at a TATA promoter may be different from those at an Inr promoter, as suggested previously (Zenzie-Gregory et al. 1992). If an activator influences a step that is rate limiting at only one core promoter class, it would exhibit a preference for that class, even if it does not directly communicate with selectivity factors like NC2 or TIC-2/3. Although most biochemical parameters that have been studied are similar among various core promoter classes, a few differences have been noted (for review, see Smale et al. 1998). One difference is that the reinitiation of transcription appears to be more efficient with TATA-containing promoters than with TATA-less promoters (Yean and Gralla 1997). An activator that selectively enhances the reinitiation frequency may therefore increase the strength of one core promoter class. An alternative possibility is that the conformation of the TFIID complex may differ when it is bound to different core promoter classes, perhaps exposing different surfaces that make the complex competent for activation by distinct subsets of transcriptional activators (e.g., Knutson et al. 2000). A better understanding of the differences between transcription initiation from different classes of promoter, and of the basic mechanisms of transcriptional activation, will be required for a complete understanding of the mechanistic basis of core promoter preferences.

Summary
The studies cited above provide strong evidence that core promoter diversity is an important contributor to combinatorial regulation. In the future, it will be important to subject this hypothesis to more stringent tests. With respect to the study by Butler and Kadonaga (2001) in this issue, it will be important to identify the enhancers that exhibit core promoter preferences, as well as the promoters that may be relevant targets of those enhancers. Evidence that the endogenous core promoters possess the anticipated structures would provide considerable support for the hypothesis. In addition to confirming the importance of core promoter preferences for combinatorial regulation, it will be important to explore in greater depth the mechanistic basis of these preferences. In some respects, this goal will be difficult to achieve until current controversies regarding the basic mechanisms of transcriptional activation have been resolved. On the other hand, because the core promoter preferences of transcriptional activators lead to a number of testable predictions, further exploration of the mechanisms underlying these preferences may contribute to the resolution of the controversies.


extfieldsofstudy: Biology Medicine

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THIK-1 and THIK-2, a Novel Subfamily of Tandem Pore Domain K+ Channels* 210

Two cDNAs encoding novel K+channels, THIK-1 and THIK-2 (tandem pore domainhalothane inhibited K +channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K+ channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1–14q24.3 (human (h) THIK-1) and 2p22–2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K+channel displaying weak inward rectification in symmetrical K+ solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.

Two cDNAs encoding novel K ؉ channels, THIK-1 and THIK-2 (tandem pore domain halothane inhibited K ؉ channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K ؉ channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1-14q24.3 (human (h) THIK-1) and 2p22-2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K ؉ channel displaying weak inward rectification in symmetrical K ؉ solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.
Relatively little is known about the function of 2P K ϩ channels in vivo. Recently, endogenous currents with properties similar to those of TASK channels have been found in the heart (17), in arterial chemoreceptor cells (18), in zona glomerulosa cells of the adrenal cortex (19), in cerebellar granular cells (20), and in motoneurons (21). TASK-1 currents have been shown to be coupled to the activation of thyrotropin-releasing hormone receptor 1 (TRH-R1; Ref. 21) and angiotensin II receptors (AT1a; Ref. 19); thus, TASK-1 channels are regulated also by mechanisms other than extracellular pH. Furthermore, another member of this subfamily, TASK-3, is activated by depolarization and modulated by extracellular divalent cations (5).
With TREK-1, TREK-2, and TRAAK, three mechanosensitive channels have been cloned (10,22). Other factors that have been reported to modulate the activity of these mechanosensitive channels include heat (TREK-1; Ref. 23 25). The third subfamily of 2P K ϩ channels so far comprises TWIK-1 and TWIK-2, both of which are expressed in multiple tissues (12,13,15). TWIK-1 shows weak inward rectification with symmetrical K ϩ concentrations (12). 2 The current carried by TWIK-2 is heat-sensitive and shows rapid time-dependent inactivation at 37°C (15). Both channels may contribute to setting the resting membrane potential, but their specific function in various tissues is not yet clear.
In this paper we describe the cloning of the first two members of a novel subfamily of 2P K ϩ channels. One of these channels, rTHIK-1, was found to be expressed in all tissues tested. Heterologous expression of rTHIK-1 in Xenopus oocytes induced a current that could be activated by arachidonic acid and inhibited by the volatile anesthetic halothane. The second novel 2P K ϩ channel, THIK-2, is closely related to THIK-1 (58% identity at the amino acid level) but could not be functionally expressed. THIK-2 was strongly expressed in several tissues including stomach, liver, and kidney and was particularly abundant in the brain.

EXPERIMENTAL PROCEDURES
Data Base Analysis-BLAST searches of the expressed sequence tag data base (dbEST), the genomic survey sequence data base (dbGSS), and the human genomic data base (htgs) identified several human and rat EST clones, one human GSS clone, and three human genomic data base entries of novel 2P K ϩ channel subunits. One rat EST clone (GenBank TM accession number AI070460) and one human GSS clone (GenBank TM accession number AQ898820) were purchased from Research Genetics (Huntsville, AL) and completely sequenced.
Isolation of Rat THIK-1 and THIK-2 cDNAs-About 1 ϫ 10 6 clones of a -ZAPII rat brain or a -ZAPII rat heart cDNA library (Stratagene) plated with XL1-Blue MRFЈ cells were screened with Digoxigeninlabeled THIK-2 and THIK-1 fragments derived from the EST or GSS clones, respectively, and positive clones were detected using CSPD (Roche Molecular Biochemicals) as chemoluminescence substrate. After purification of isolated plaques by two further screenings, pBSKϩ plasmids containing the cDNAs were excised from -clones and sequenced.
Sequence Analysis-For sequence analysis, the GCG program package implemented at the Heidelberg Unix Sequence Analysis Resources (HUSAR) was used. The program PROSITE was used for identification of sequence consensus sites, the program GAP for pairwise sequence alignments, the program CLUSTAL W for creation of multiple alignments, and the program CLUSTREE for computation of the phylogenetic tree. Standard default parameters were used for the calculations.
Electrophysiological Analysis-cDNAs encoding rTHIK-2, rTHIK-1, and chimeras between the two were cloned into the expression vector pSGEM (a gift of Dr. M. Hollmann) for expression in Xenopus laevis oocytes. Capped run-off poly(A ϩ ) cRNA transcripts were synthesized and injected individually or in combination into defolliculated oocytes at constant amounts (ϳ3 ng each). Oocytes were incubated at 19°C in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5 mM HEPES, pH 7.4 -7.5), supplemented with 100 g/ml gentamicin and 2.5 mM sodium pyruvate, and assayed 48 h after injection. Two-electrode voltage-clamp measurements were performed with a Turbo Tec-10 C amplifier (npi, Tamm, Germany), which has a settling time of 100 s for a 100-mV voltage pulse. Data were recorded via an EPC9 (Heka Electronics, Lambrecht, Germany) interface at sampling rates up to 20 kHz using Pulse/Pulsefit software (Heka). The Oocytes were placed in a small-volume perfusion chamber and bathed with ND96 or "high K ϩ " solution (96 mM KCl, 2 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5 mM HEPES, pH 7.4 -7.5).
In Situ Hybridization-Wistar rats were decapitated under ether anesthesia, and their brains were removed and frozen on powdered dry ice. Tissue was stored at Ϫ20°C until cutting. Sixteen-m sections were cut on a cryostat, thaw-mounted onto silane-coated slides, and airdried. After fixation for 10 min in 4% paraformaldehyde dissolved in PBS, slides were washed in PBS, dehydrated, and stored in ethanol until hybridization.

RESULTS
Cloning of Two Novel 2P K ϩ Channel cDNAs-Two cDNA populations coding for novel 2P K ϩ channels were isolated from two rat cDNA libraries. The first channel was isolated from a rat brain and a rat heart library and was named THIK-1 for tandem pore domain halothane inhibited K ϩ channel. The second, closely related channel was found only in the rat brain library and was named THIK-2 despite the fact that it was not functional when expressed in Xenopus oocytes. Sequence analysis of the isolated cDNA clones revealed complete open reading frames ( Fig. 1) for rTHIK-1 (2032 bp) and rTHIK-2 (1900 bp). The nucleotide sequences predict proteins of 405 (rTHIK-1) and 430 (rTHIK-2) amino acids, respectively, which show the typical features of 2P K ϩ channels: four transmembrane regions, two pore-forming regions, a large extracellular linker between M1 and P1, a short N terminus and a larger C terminus. As indicated in Fig. 1, A and B, several putative intracellular phosphorylation sites for protein kinase A, protein kinase C, and casein kinase were found in both sequences. In the M1-P1 linker region, rTHIK-1 harbors two putative glycosylation sites (N-{P}-[S/T]-{P}), whereas rTHIK-1 possesses only one. The rTHIK-2 protein is significantly longer at the N terminus, including three putative protein kinase C phosphorylation sites and several repetitive amino acid elements.
Sequence comparison identified the human orthologs of THIK-2 in data base entry AC009600 and of THIK-1 in two overlapping data base entries (AL355074, AL137128). In accordance with the human nomenclature committee (HUGO), the two novel channel genes were named KCNK12 (THIK-2) and KCNK13 (THIK-1), respectively. The human channel subunits are 98% (hTHIK-2) and 84% (hTHIK-1), identical to their rat orthologs. hTHIK-1 and hTHIK-2 are 61.8% identical and 67.5% similar to each other, whereas only about 25-35% identity was found to other 2P K ϩ channels (Fig. 2B). Interestingly, the intracellular M2-M3 linker was 16 -20 amino acids longer than that of other cloned 2P K ϩ channels. The cytosolic Cterminal domains of the THIK channels are 57% identical to each other (see supplemental material), whereas no significant homology was found to other C-terminal domains of 2P K ϩ channel. The pore regions of both THIK channels are nearly identical (no mismatch in a stretch of 13 amino acids in the first pore region and only one mismatch in the second pore), whereas there is considerable difference to other 2P K ϩ channels (Fig. 2C). From the homology scores, the unique structural features of THIK channels and the phylogenetic clustering ( Fig. 2A), it is obvious that THIK 1 (KCNK13) and THIK-2 (KCNK12) form a novel subfamily among the 2P K ϩ channels. Supplemental material (multiple sequence alignment excluding the unrelated C-terminal domains and pairwise alignment of THIK-1 and THIK-2 C-terminal domains) is available in the on-line version.
Analysis of the entries of the human genome databases showed that both genes described here have a similar structure, with a single large intron of 48.1 kilobases (KCNK12) and 121.5 kilobases (KCNK13), respectively, splitting the coding region at the first pore GYG motif. This intron is conserved in all mammalian 2P K ϩ channel genes (KCNK1-10) cloned so far. Chromosomal localization assigned KCNK12 to the human chromosomal region 2p22-2p21 between markers WI-10633 and SGC34238. KCNK13 was assigned to chromosomal region 14q24.1-14q24.3 between markers IB3608 and SGC30527, which is very close to the human TREK-2 (KCNK10) gene. The results of the chromosomal assignments are summarized in Table I. The tissue distribution of the rTHIK-1 and rTHIK-2 was obtained by reverse transcription-PCR analysis. Intronspanning primers were used to avoid false positive results due to genomic contamination. rTHIK-1-specific products were amplified from all tissues tested. In contrast, rTHIK-2 expression was found in brain, lung, kidney, liver, stomach, and spleen but not in skeletal muscle, heart, and testis (Fig. 3). In addition, putative glycosylation sites (h), protein kinase A phosphorylation sites (a),protein kinase C phosphorylation sites (1) and casein kinase phosphorylation sites (⌬) are indicated below the amino acid sequence. Consensus sites were identified using the program PROSITE.
In Situ Hybridization of Rat Brain-The mRNA distribution of rTHIK-1 and rTHIK-2 in the adult rat brain as detected by in situ hybridization is highly differential, with little overlap (summarized in Table II). Two different antisense probes were used for either subunit. The resulting labeling profiles were identical for each subunit, indicating that the probes were specific. rTHIK-2 mRNA was found to be widely expressed in most brain regions, with highest levels in the cerebellar granule cell layer, the mitral and granule cell layers of the olfactory bulb (Fig. 4E), and in the anterodorsal and anteroventral nuclei of the thalamus (Fig. 4F). Strong signals were also found in the thalamic ventral posterior nuclei (Fig. 4G), cortex, hippocampus, the pontine nucleus, the red nucleus (Fig. 4E), the oculomotor nucleus, and some nuclei of the amygdala. In the brainstem, elevated levels were found in all trigeminal sensory nuclei, in the tegmental and reticular nuclei, and in the ventral cochlear nuclei. rTHIK-2 mRNA was also found in some non-neuronal cells such as the ependymal lining of the ventricles. rTHIK-2 was found to be absent from the substantia nigra, gracile nuclei, inferior olive, and from most parts of the caudate putamen and septal nuclei as well as all white matter pathways.
In contrast, rTHIK-1 mRNA expression was found to be rather weak and restricted to only a few brain regions and nuclei (Fig. 4, A-D). Substantial expression levels were detected only in the granule cell layer of the olfactory bulb, in the olfactory tubercle, the lateral septum (Fig. 4A), and in distinct hypothalamic and thalamic nuclei (ventromedial hypothalamic nucleus, lateral mamillary nucleus, reticular nucleus, reunions nuclei (Fig. 4, B and C)). Other rTHIK-1-positive structures were only partially labeled. The cortex, for example, was positive only in layer II, the striatum was labeled only in the caudal part, and the dentate gyrus granule cell layer of the dentate gyrus exhibited particularly high mRNA levels in the caudalventral part (Fig. 4D). Similarly, within a nuclear group, only specific subnuclei were positive, e.g. the dorsal subnucleus of the lateral septum (Fig. 4A), the magnocellular part of the red nucleus (Fig. 4D), a subnucleus of the lateral habenula (Fig. 4,  A and C), one lateral parabrachial subnucleus, and the ventral nucleus of the cochlear nuclei.
Heterologous Expression in Xenopus Oocytes-At day 2 after injection of rTHIK-1 cRNA into Xenopus oocytes, large currents were recorded using the two-electrode voltage clamp (Fig. 5A), which were not seen under control conditions. The steady-state amplitude of the currents measured at ϩ60 mV was 21.85 Ϯ 4.45 A (n ϭ 7) in the presence of 2 mM external K ϩ . Current activation in response to depolarizing voltage steps was not FIG. 2. Sequence comparison of eleven 2P K ؉ channels. A, phylogenetic tree. The GenBank TM accession numbers for the different channels are: hTHIK-1, AF287303; hTHIK-2, AF287302; hTASK-1, AF006823; hTASK-3, AF212829; hTREK-1, AF129399; rTREK-2, AF196965; hTRAAK, AF247042; hTWIK-1, U33632; hTWIK-2, AF117708; KCNK7, AF110522; and mTASK-2, AF084830. The CLUSTAL/CLUSTREE algorithm (Heidelberg UNIX Sequence Analysis Resources) was used for computation. B, pairwise sequence identity scores of THIK channels. The program GAP was used for calculation. C, multiple sequence alignment of the two pore regions of the 2P K ϩ channels. Conserved residues are shown in bold, and the only residue different in THIK-1 and THIK-2 pore domains is labeled with an asterisk. Additional information on sequence comparison (multiple alignment excluding C-terminal domains of the above mentioned channels, pairwise alignment of THIK-1 and THIK-2 C-terminal domains) is available on the on-line version. instantaneous but, when fitted to a single exponential, showed an activation time constant of 0.97 Ϯ 0.25 ms at ϩ60 mV. During a 500-ms voltage pulse, currents did not inactivate. The steady-state current-voltage relationship of rTHIK-1 showed only weak voltage dependence. With low external K ϩ , moderate outward rectification was observed, whereas with nearly symmetrical K ϩ , weak inward rectification was found (Fig. 5B). When external K ϩ was altered to examine the ion selectivity of rTHIK-1, the measured reversal potentials were very close to the K ϩ equilibrium potential (calculated on the assumption of an intracellular K ϩ concentration of 100 mM). The linear regression fit to the data gave a slope of Ϫ53 mV/decade (Fig. 5C), as would be expected for highly selective K ϩ channels. THIK-1 could be partially blocked by Ba 2ϩ ions; application of 1 mM Ba 2ϩ inhibited the instantaneous outward current measured at ϩ60 mV (in the presence of 2 mM external K ϩ ) by about 60% (n ϭ 4, not shown).
In contrast, the currents measured in Xenopus oocytes after injection of rTHIK-2 (Fig. 5D) were not significantly different from those of noninjected or water-injected oocytes (n ϭ 10). To test whether both channel proteins were translated and targeted to the surface membrane, rTHIK-1 and rTHIK-2 were tagged with EGFP at the N terminus. 48 h after injection of EGFP-rTHIK-2, confocal microscopy showed strong membrane fluorescence that was very similar to that found after injection of EGFP-rTHIK-1 (Fig. 6A). Thus rTHIK-2 subunits appear to be targeted to the outer membrane. The reason for the lack of functional expression of rTHIK-2 was further studied by constructing chimeric subunits in which the first part of the rTHIK-2-coding region (M1-P1-M2) was fused to the second half of rTHIK-1 (M3-P2-M4) and vice versa. Injection of the chimeric cRNAs did not induce any current in Xenopus oocytes.
Some voltage-activated (Kv) or inwardly rectifying (Kir) K ϩ channel subunits that do not functionally express as homomers can modify the activity of other subunits by heteromerization. Therefore we investigated the possibility that rTHIK-1 and rTHIK-2 subunits might coassemble to form heterodimeric channels. When rTHIK-1 and rTHIK-2 cRNAs were injected at equimolar amounts (n ϭ 5), all macroscopic current properties were virtually indistinguishable from rTHIK-1 currents, and current amplitudes were not significantly different (Fig. 6, B and C). Furthermore, the currents induced by injection of rTHIK-1 were unaffected by injection of larger amounts of rTHIK-2 cRNAs (ratio 1:5). The apparent absence of heteromerization of rTHIK-2 and rTHIK-1 subunits suggests that rTHIK-2 is not a regulatory subunit of the rTHIK-1 conductance.
Regulation of rTHIK-1 Channels-To test for functional similarities to other members of the two pore domain K ϩ channel family, we studied the modulation of rTHIK-1 channel activity by various experimental interventions such as extracellular acidification, application of the polyunsaturated fatty acid arachidonic acid, or application of the volatile anesthetic halothane (Fig. 7A). The outward current carried by rTHIK-1 was FIG. 3. Tissue distribution of rTHIK-1 and rTHIK-2. cDNA from the tissues indicated was analyzed for the presence of a 326-bp fragment of rTHIK-1 or a 250-bp fragment of rTHIK-2, indicated as a white arrow. Note that both primer pairs were intron-spanning to exclude possible genomic contamination. A pBR322/HaeIII marker was used in the left-most lane.

TABLE II
Distribution of rTHIK-1 and rTHIK-2 mRNA in the adult rat brain In situ hybridization signals obtained for 33 P-labeled oligonucleotide probes on adult rat brain sections were rated according to the relative grain density: ϩϩϩϩ, very abundant; ϩϩϩ, abundant; ϩϩ, moderate; ϩ, low; Ϯ, just above background; 0, no expression. Note that only selected brain regions with elevated expression levels or markedly differential expression patterns are included in the table. AHiPM, amygdalohippocampal area, posteromedial part; PMCo; posteromedial cortical amygdaloid nucleus; Mo5, -7, -12, motor nuclei of cranial nerves 5, 7, and 12. only weakly inhibited by extracellular acidification to pH 6, which is in marked contrast to the pronounced pH sensitivity of TASK-1 (KCNK3; half-maximal block at pH 7.38), as illustrated in Fig. 7B. Lowering the pH to 4.5 inhibited the outward current carried by rTHIK-1 by 34 Ϯ 8% (n ϭ 5). Similar to TASK-3, proton block of rTHIK-1 occurred with a fast time course (Fig. 7A) and was independent of the membrane potential (Fig. 7B). The sensitivity of rTHIK-1 to intracellular pH was tested by the "rebound acidification" technique. 20 mM NH 4 Cl was applied for 5 min and then removed, which should decrease the intracellular pH by about 1 unit. This intervention produced no measurable change in the current carried by rTHIK-1 (n ϭ 3), indicating that rTHIK-1 is not modulated by intracellular pH.
Since TREK-1 has been reported to be heat-sensitive (23), we also studied the temperature dependence of rTHIK-1. Raising the temperature from 22 to 37°C increased the current amplitude by a factor of 1.6 (n ϭ 3), in agreement with van't Hoff's rule, whereas TREK-1 is augmented by a factor of 10 under the same conditions (23). These findings suggest that, unlike TREK-1, rTHIK-1 is not a heat-sensitive channel. Lysophosphatidylcholine (3 M) has also been shown to activate TREK-1 and TRAAK (24). Application of 10 M lysophosphatidylcholine to Xenopus oocytes expressing rTHIK-1 induced only a very minor (up to 20%) increase in current amplitude, which may be attributable to a nonspecific effect of the lysophospholipid.
Application of arachidonic acid to the bath solution induced a rapid increase in the outward current carried by rTHIK-1 channels (Fig. 7A). This effect could be washed out within 5 min. In the presence of 5 M arachidonic acid, the current was increased by 85 Ϯ 24% (n ϭ 5) at ϩ30 mV. As can be seen from Fig. 7C, the current activated by arachidonic acid was outwardly rectifying. It reversed at the calculated potassium equilibrium potential when the external K ϩ concentration was altered. The concentration dependence of the effect of arachidonic acid on rTHIK-1 could be described by a K d of 0.98 M and a Hill coefficient of 1.97 (Fig. 7C). The effects of arachidonic acid reported here are similar to the effects found in TREK-1 (KCNK2) and TRAAK (KCNK4) (7,9,24).
Two of the known 2P K ϩ channels, TREK-1 and TASK-1, are activated by the volatile anesthetic halothane (27). Surprisingly, our whole-cell recordings in Xenopus oocytes showed that the current carried by rTHIK-1 was rapidly and reversibly inhibited by halothane (Fig. 7, A and D). When the membrane potential was held at ϩ30 mV, application of 5 mM halothane reduced rTHIK-1 currents by 56 Ϯ 5% (n ϭ 5). The fit of the concentration-effect curve gave a K d of 2.83 mM and a Hill coefficient of 1.06. Under the same experimental conditions, application of chloroform (1 mM) had no effect (n ϭ 3; data not shown). DISCUSSION THIK-1 and THIK-2 are the first two members of a novel 2P K ϩ channel subfamily. Although they show the typical features of other 2P K ϩ channels, such as four transmembrane regions, two pore-forming regions, and a large extracellular M1-P1 linker region, the two novel channels are only about 25-35% identical to the other known 2P K ϩ channels but 61.8% identical to each other. One notable difference is the larger cytosolic M2-M3 linker region, containing three (rTHIK-2) or one (rTHIK-1) putative phosphorylation site(s). In addition, the THIK-2 protein has an unusual N terminus containing several repeats of proline, arginine, and cysteine residues. The Cterminal domain of THIK-1 and THIK-2 shows no significant homology to other mammalian 2P K ϩ channels.
The reversal potential of the current induced by injection of rTHIK-1 cRNA in Xenopus oocytes followed the calculated K ϩ equilibrium potential when external K ϩ was changed. This suggests that the THIK channels are mainly permeable to K ϩ ions. The whole-cell current induced by heterologous expres- FIG. 5. Heterologous expression of rTHIK-1 and rTHIK-2 in Xenopus oocytes. Whole-cell current recordings from oocytes injected with cRNA of rTHIK-1 (A-C) and rTHIK-2 (D), respectively. A, voltage steps of 500-ms duration from a holding potential of Ϫ60 mV to potentials between Ϫ140 to ϩ60 mV. B, voltage ramps (105 mV s Ϫ1 ) from Ϫ150 to ϩ60 mV at external potassium concentrations of 2, 5, 10, 25, and 96 mM K ϩ , respectively (the holding potential preceding the ramp pulses was Ϫ60 mV). C, semi-logarithmic plot of the measured zero-current (reversal) potentials versus [K ϩ ] e . A linear regression line with a slope of 53 mV per decade was fitted to the data points. D, voltage ramps in a Xenopus oocyte injected with rTHIK-2 cRNA. The ramp protocol was identical to that shown for rTHIK-1 in panel B.

FIG. 6. Targeting of rTHIK-1 and rTHIK-2 and coexpression in
Xenopus oocytes. A, fluorescence signal of EGFP-labeled rTHIK-1 and rTHIK-2 in the oocyte membrane 48 h after cRNA injection. B, wholecell currents recorded from Xenopus oocytes after expression of rTHIK-1 alone and after coexpression of rTHIK-1 together with rTHIK-2 at RNA ratios of 1:1 and 1:5. Voltage ramps from Ϫ150 mV to ϩ60 mV were applied; the preceding holding potential was Ϫ60 mV. C, bar graph of the currents measured at ϩ60 mV. No significant difference was found between oocytes injected with rTHIK-1 alone and in combination with rTHIK-2.
sion of rTHIK-1 displayed outward rectification at physiological external K ϩ and weak inward rectification with approximately symmetrical K ϩ concentrations. It could be activated by arachidonic acid (K d , 0.98 M; Hill coefficient, 1.97) and inhibited by halothane (K d , of 2.8 mM; Hill coefficient, 1.06). Chloroform had no effect on rTHIK-1. Another 2P K ϩ channel, TWIK-2, which is almost absent in the brain, has recently been found to be inhibited by both halothane and chloroform (15). In contrast, both TREK-1 and TASK-1 are activated by halothane and isoflurane, and TREK-1 is additionally activated by chloroform and diethyl ether (27). TREK-1, TASK-1, and THIK-1 are all expressed in specific regions of the brain. The findings reported here suggest that the effects of volatile anesthetics on the brain may be more complex than hitherto assumed. We are aware of the fact that the IC 50 for the effects of halothane on THIK-1 is higher than the EC 50 for the anesthetic effects in vivo (Ϸ250 M; Ref. 28). Nevertheless, we decided to name the new channels tandem pore-domain halothane inhibited K ϩ channels because this discriminates them from some of the other 2P K ϩ channels.
Injection of rTHIK-2 cRNA in Xenopus oocytes did not produce any measurable currents. The lack of functional expression of rTHIK-2 was apparently not due to inadequate targeting, because confocal microscopy showed EGFP-tagged rTHIK-2 channels in the outer cell membrane (Fig. 6). To localize possible structural constraints in rTHIK-2 that prevent expression of functional channels, we constructed rTHIK-1/ rTHIK-2 and rTHIK-2/rTHIK-1 chimeras. However, since neither of the two chimeras was functional, the reason for the nonfunctional state of rTHIK-2 in Xenopus oocytes remains unclear. Another possibility is that rTHIK-2 might be a regulator of rTHIK-1 conductance by coassembling with rTHIK-1. This is unlikely, because whole cell currents produced by injection of rTHIK-1 cRNA were unaffected by coinjection even of 5-fold larger amounts rTHIK-2 cRNA. In situ hybridization in the brain showed little overlap between rTHIK-1 and rTHIK-2, which also argues against heteromerization. In conclusion, the high expression of rTHIK-2 in cerebral cortex, hippocampus, and olfactory bulb and the specific expression in several nuclei (Fig. 6) support the idea that rTHIK-2 is functionally important in neurons, but the available experimental evidence suggests that rTHIK-2 requires an accessory subunit or specific intracellular ligands to form a conducting pore. The strong expression in lung, kidney, and stomach suggests that rTHIK-2 may also play a role in epithelial cells.
Both human THIK genes described here have only one very large intron, 121.5 kilobases in the THIK-1 gene (KCNK13) and 48.1 kilobases in the THIK-2 gene (KCNK12), that splits the coding region of the first pore (GYG motif). In this respect, the THIK genes are similar to the TASK-3 gene (KCNK9). The genes of the TWIK and the TREK-/TRAAK families possess several introns in the coding region (10,29,30), but an intron splitting the GYG motif of the first pore region is conserved in all mammalian 2P K ϩ channel genes cloned so far and, in addition, in most of the 2P K ϩ channels of Caenorhabditis elegans (31) and Drosophila melanogaster. Another interesting feature is the colocalization of 2P K ϩ channel genes at the same chromosomal region. We have shown that THIK-1 (KCNK13) maps to the same region on the long arm of chromosome 14 as TREK-2 (KCNK10). The other 2P K ϩ channel gene pairs known so far are TRAAK (KCNK5) and KCNK7 on chromosome 11q13 (29) and TWIK-1 (KCNK1) and TREK1 (KCNK2) on chromosome 11q41-43 (32). These data are consistent with a common ancestral gene for all 2P K ϩ channels that was subject to several duplication events.
As illustrated in Fig. 2, mammalian 2P K ϩ channels can be subdivided in five subfamilies: 1) TWIK/KCNK7, 2) TREK/ TRAAK, 3) TASK-1/TASK-3, 4) THIK, and 5) TASK-2. These channels display a wide variety of electrophysiological and regulatory characteristics that are usually not confined to one of the subfamilies. The steady-state current voltage relation measured in the whole-cell configuration at symmetrical K ϩ concentrations was found to be inwardly rectifying (TWIK-1, TWIK-2, and THIK-1), outwardly rectifying (TREK-1, TASK-3), or linear (TREK-1, TASK-1, TRAAK). Some of the 2P K ϩ channels show a pronounced sensitivity to intracellular (TREK-1) or extracellular pH (TASK-1, TASK-2, TASK-3). The pharmacological profile of the 2P K ϩ channels is also very diverse and not related to subfamilies. Some 2P K ϩ channels are activated by volatile anesthetics (TREK-1, TASK-1); other channels are inhibited (TWIK-2, THIK-1). Some channels are activated by fatty acids such as arachidonic acid (TREK-1, TRAAK, TWIK-2, THIK-1) or by phospholipids such as lysophosphatidylcholine (TREK and TRAAK). In addition, some of the 2P K ϩ channels are mechanosensitive (TREK-1, TREK-2, and TRAAK) or heat-sensitive (TREK-1). The most remarkable common characteristic of all 2P K ϩ channels known so far is that their regulation by physical and chemical stimuli is very complex. The difficulty in identifying their function is probably related to this complex regulation, which needs to be studied in the native cells in which the channels are expressed.


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Regulation of interleukin (IL)-18 receptor alpha chain expression on CD4(+) T cells during T helper (Th)1/Th2 differentiation. Critical downregulatory role of IL-4.

Interleukin (IL)-18 has been well characterized as a costimulatory factor for the induction of IL-12–mediated interferon (IFN)-γ production by T helper (Th)1 cells, but also can induce IL-4 production and thus facilitate the differentiation of Th2 cells. To determine the mechanisms by which IL-18 might regulate these diametrically distinct immune responses, we have analyzed the role of cytokines in the regulation of IL-18 receptor α chain (IL-18Rα) expression. The majority of peripheral CD4+ T cells constitutively expressed the IL-18Rα. Upon antigen stimulation in the presence of IL-12, marked enhancement of IL-18Rα expression was observed. IL-12–mediated upregulation of IL-18Rα required IFN-γ. Activated CD4+ T cells that expressed low levels of IL-18Rα could produce IFN-γ when stimulated with the combination of IL-12 and IL-18, while CD4+ cells which expressed high levels of IL-18Rα could respond to IL-18 alone. In contrast, T cell stimulation in the presence of IL-4 resulted in a downregulation of IL-18Rα expression. Both IL-4−/− and signal transducer and activator of transcription (Stat)6−/− T cells expressed higher levels of IL-18Rα after TCR stimulation. Furthermore, activated T cells from Stat6−/− mice produced more IFN-γ in response to IL-18 than wild-type controls. Thus, positive/negative regulation of the IL-18Rα by the major inductive cytokines (IL-12 and IL-4) determines the capacity of IL-18 to polarize an immune response.

The IL-18R complex is composed of two known chains, IL-18 receptor ␣ chain (IL-18R ␣ ) * and IL-18R ␤ (15)(16)(17)(18)(19)(20). IL-18R ␣ (IL-1R5) was originally described as IL-1 receptor-related protein (IL-1Rrp) because of its homology with the IL-1/Toll receptor family and is the extracellular binding domain of the IL-18R complex. IL-18R ␤ (also known as IL-18RAcPL or IL-1R7) is the signal transducing chain of the IL-18R complex and is also a member of the IL-1R family. It has been widely believed that the IL-18R ␣ is not expressed on naive T cells, but is induced during differentiation of Th1, but not Th2, cells. Here, we have used a recently developed polyclonal antibody to IL-18R ␣ to investigate regulation of its expression on differentiating CD4 ϩ T cells. In contrast to previous studies, we demonstrate that resting peripheral CD4 ϩ and CD8 ϩ T cells express IL-18R ␣ . Although it has been proposed that IL-12 alone may upregulate expression of the IL-18R ␣ , culture of naive CD4 ϩ T cells in IL-12 or IL-18 alone had no effect on IL-18R ␣ expression. However, activation of T cells via the TCR in the presence of IL-12, but not IL-18, resulted in marked upregulation of IL-18R ␣ expression. The combination of IL-18 and IL-12 further enhanced IL-18R ␣ expression during TCR activation. Although IFN-␥ did not directly regulate IL-18R ␣ expression, IL-12-mediated upregulation of IL-18R ␣ was not observed in the absence of IFN-␥ . After activation in the presence of IL-12, the capacity of CD4 ϩ T cells to produce IFN-␥ in response to IL-18 alone correlated with their expression of high levels of the IL-18R ␣ . In sharp contrast, the combination of TCR ligation and IL-4 led to a marked downregulation of IL-18R ␣ on unprimed CD4 ϩ T cells. The downregulatory effects of IL-4 on IL-18R ␣ were completely dependent on expression of signal transducer and activator of transcription (Stat)6. Collectively, these results demonstrate that IL-18R ␣ expression is finely regulated during the course of CD4 ϩ T cell differentiation along either the Th1 or Th2 pathways. They are most consistent with a model in which IL-12/IFN-␥ play a critical function in upregulation of IL-18R ␣ expression during differentiation along the Th1 pathway, while IL-4 plays a downregulatory role on IL-18R ␣ during Th2 differentiation. These data are critical to furthering our understanding of the proposed dual role of IL-18 as a differentiation factor for both Th1 and Th2 responses as well as its possible therapeutic use as an adjuvant.

Materials and Methods
Animals. Female C57BL/6, C57BL/6 IFN-␥ Ϫ / Ϫ , C57BL/6 IL-4 Ϫ / Ϫ mice, and 5CC7 B10.A/Ai recombination activating gene ( RAG)-2 Ϫ / Ϫ mice expressing a TCR transgene specific for cytochrome C were obtained from Taconic Farms and used at 4-8 wk of age. Female BALB/c and Stat6 Ϫ / Ϫ (C129S2-Stat6) mice were obtained from Jackson ImmunoResearch Laboratories. All animals were housed under specific pathogen-free conditions and provided food and water ad libitum. Animals were maintained according to National Institutes of Health Animal Care Guidelines.
Flow Cytometry. Cells were washed twice in PBS, resuspended in PBS, and placed (1-2 ϫ 10 6 cells) into individual wells of a 96-well V-bottomed Costar plate. The cells were first incubated for 5 min at 4 Њ C with 1 g of rat anti-mouse CD16/CD32 (Fc block; BD PharMingen) to block nonspecific binding of goat Ig/anti-IL-18R ␣ antibody to Fc receptors. Cells were then incubated with either 1 g of goat IgG or 1 g of goat anti-murine IL-18R␣ (R&D Systems) diluted in PBS containing 5% normal mouse serum (PBS-NMS). Anti-IL-18R␣ is an affinity-purified, polyclonal goat-specific IgG antibody that recognizes the extracellular domain (peptide 19-326) of recombinant murine IL-18R␣. Cells were incubated with the respective antibody for 30 min, 4ЊC, washed two times with PBS, incubated in PBS-NMS for 10 min, and then stained with SP-biotinylated mouse antigoat IgG (H plus L) diluted in PBS-NMS (Jackson ImmunoResearch Laboratories) for 20 min. Cells were subsequently washed two times in PBS and then resuspended in streptavidin-PE (BD PharMingen) diluted in PBS. After a 10-min incubation at 4ЊC, FITC-labeled anti-CD4 (BD PharMingen) was added directly to wells for an additional 20-min incubation at 4ЊC. Cells were again washed two times in PBS and resuspended in PBS. 7-AAD was added immediately before FACS ® analysis to exclude dead cells from analysis. Analysis was performed using CELLQuest™ software (Becton Dickinson).
Data are presented as the difference (⌬) between the mean fluorescence intensity (MFI) of the positive stain (anti-IL-18R␣) and the MFI of the negative control (goat Ig).
Cytokine Assays. T cells that had been primed in vitro for 4-7 d under various polarizing conditions were resuspended in fresh IL-2 medium. Cells (10 6 cells per milliliter) were cultured in 24-well plates for 48 h with 10 ng/ml IL-12, 30 ng/ml IL-18, or both. Cell-free supernatants were collected and levels of IFN-␥ determined by ELISA (R&D Systems). Alternatively, primed cells were restimulated with either immobilized anti-CD3 (5 g/ml 2C11; BD PharMingen) or IL-12 and IL-18 (as above) for 6-8 h, and monensin was added for the final 4 h of culture. Cells were harvested, permeabilized with saponin/PBS buffer, then stained for intracellular IFN-␥ and IL-4.

Results
Naive T Cells Express the IL-18R␣. In contrast to previous studies which only detected the IL-18R␣ on fully differentiated Th1 cells (22), a majority (82 Ϯ 2%) of both CD4 ϩ and CD8 ϩ splenic T cells from C57BL/6 mice were reactive by FACS ® analysis with the polyclonal antibody to recombinant IL-18R␣ (Fig. 1, A and C). In multiple experiments (n ϭ 5) of this type the ⌬MFI of IL-18R␣ expression on freshly explanted CD4 ϩ T cells was 143 Ϯ 11. CD4 ϩ and CD8 ϩ T cells in lymph node expressed levels of IL-18R␣ similar to that seen in spleen (data not shown). We also detected high levels of IL-18R␣ on mature CD4 ϩ and CD8 ϩ single positive T cells in the thymus, while CD4 ϩ CD8 ϩ double positive thymocytes expressed rela-tively low levels (data not shown). Thus, expression of the IL-18R␣ appears to be acquired during the process of T cell differentiation in the thymus. Constitutive expression of the IL-18R␣ was independent of either IL-12 or IFN-␥ as the level of expression of the IL-18R␣ on CD4 ϩ cells from IL-12 Ϫ/Ϫ or IFN-␥ Ϫ/Ϫ mice was identical to CD4 ϩ T cells from wild-type mice (data not shown). Analysis of other cell types revealed low levels of IL-18R␣ expression on B220 ϩ splenic B cells ( Fig. 1 D), while the highest level of IL-18R␣ expression was found on NK1.1 ϩ cells (Fig. 1  E). We consistently identified a small population )%01-6ف( of splenic CD4 ϩ cells that expressed high levels of the IL-18R␣ ( Fig. 1 A, M2, MFI 1,072 Ϯ 286). This IL-18R␣ hi population was absent in the spleens of 5CC7 TCR transgenic mice on a RAG-2 Ϫ/Ϫ background ( Fig. 1 B, arrow), suggesting the CD4 ϩ IL-18R␣ hi population in the former could be a population of previously activated/memory T cells or CD4 ϩ NK1.1 ϩ T cells. Further analyses revealed the majority )%09ف( of CD4 ϩ IL-18R␣ hi cells are NK1.1 Ϫ and express markers typically associated with memory/activated T cells (data not shown). Thus, two distinct levels of IL-18R␣ expression (IL-18R␣ int and IL-18R␣ hi ) can be detected on CD4 ϩ T cells in spleens from normal animals.
Stimulation with IL-12 or IL-18 Alone Does Not Lead to Upregulation of IL-18R␣ on CD4 ϩ IL-18R␣ int T Cells. Previous reports have suggested that IL-12 in the absence of TCR stimulation was capable of inducing IL-18R␣ expression, thus enabling IL-18 to synergize with IL-12 to enhance IFN-␥ production (23). To evaluate the effects of cytokines on IL-18R␣ expression, we cultured spleen cells from normal C57BL/6 mice for 96 h in IL-2-containing media alone or in IL-2-containing media supplemented with either IL-12 or IL-18. Preliminary studies demonstrated that IL-2 was required to maintain adequate viability of the cells. Culture with IL-2 alone led to a decrease in IL-18R␣ expression on CD4 ϩ IL-18R␣ int cells ( Fig. 1  . Thus, the primary effects of IL-12 on freshly explanted CD4 ϩ T cells from normal animals appear to be via upregulation of the IL-18R␣ on the minor population of CD4 ϩ T cells that already express high levels of this receptor and which appear to be memory/activated T cells.

IL-12 in Concert with TCR Stimulation Induces Upregulation of IL-18R␣ Expression on CD4 ϩ T Cells.
To examine the effects of TCR stimulation on IL-18R␣ expression, CD4 ϩ T cells from naive C57BL/6 mice were stimulated for 2 d with immobilized anti-CD3 in IL-2-containing media, with or without IL-12/IL-18. After 2 d in culture, the cells were harvested, washed, and placed into fresh IL-2 media with fresh cytokines for an additional 2 d. This twostep culture system was used because significant apoptosis was observed when T cells were cultured for 4 d on platebound anti-CD3. IL-18R␣ expression was detected on CD4 ϩ T cells stimulated with anti-CD3 and IL-2, but the level of expression was consistently less than that observed on freshly explanted CD4 ϩ T cells (compare Fig. 3 A with Fig. 1 A) and CD4 ϩ T cells cultured in IL-2 alone (compare Fig. 2 A, M1 with Figs. 3 A and 1 A). However, the addition of IL-12 to anti-CD3-stimulated cultures led to marked upregulation of IL-18R␣ expression (Fig. 3 B), while the addition of IL-18 alone did not result in enhanced IL-18R␣ expression (Fig. 3 C). IL-18 did synergize with IL-12 to enhance IL-18R␣ expression (Fig. 3 D).
Resting CD4 ϩ T cells failed to produce IFN-␥ on exposure to IL-12, IL-18, or the combination of IL-12/IL-18 (data not shown). However, after anti-CD3 induced T cell activation, large amounts of IFN-␥ were produced in response to IL-12/IL-18 irrespective of the priming conditions (Fig. 3, E-H). More importantly, the upregulation of IL-18R␣ expression seen when T cells were cultured with IL-12 correlated with the ability of IL-18 to induce IFN-␥ production in the absence of IL-12 ( Regulation of IL-18R␣ Expression by IFN-␥. As IFN-␥ is induced by IL-12, it was possible that IL-12-mediated upregulation of the IL-18R␣ was secondary to IFN-␥ production. To test this, we stimulated T cells from IFN-␥ Ϫ/Ϫ mice with anti-CD3 and IL-2. After 4 d of culture, CD4 ϩ T cells from IFN-␥ Ϫ/Ϫ mice expressed levels of IL-18R␣ that were similar to wild-type C57BL/6 mice (Fig.  4, A and B). Therefore, the mere absence of IFN-␥ does not lead to downregulation of IL-18R␣ expression. However, IL-12-dependent upregulation of IL-18R␣ expression was severely impaired in the absence of IFN-␥. A majority of CD4 ϩ T cells from IFN-␥ Ϫ/Ϫ mice failed to upregulate IL-18R␣ expression when cultured with anti-CD3 and IL-12 (M1; Fig. 4, C and D). Addition of IFN-␥ to these cultures restored IL-12-driven upregulation of  IL-18R␣; however, IFN-␥ itself did not enhance IL-18R␣ expression in the absence of IL-12 (M1; Fig. 4, E and F). These data demonstrate that IFN-␥ is required for IL-12mediated upregulation of IL-18R␣ expression and most likely functions by inducing/maintaining IL-12R␤2 expression. However, we consistently observed a minor population of T cells that could upregulate their IL-18R␣ in an IL-12-dependent but IFN-␥-independent fashion (M2; Fig. 4, B and D-F). We suspect these are memory T cells, based on our observations that memory cells can upregulate their IL-18R␣ expression in response to IL-12 alone (Fig. 2, A-D).
IL-4 in Concert with TCR Stimulation Induces Downregulation of IL-18R␣. While previous studies have focused on IL-12 and its ability to augment IL-18 responses, very little is known about the cytokines which may negatively regulate IL-18R␣ expression. Therefore, we stimulated C57BL/6 CD4 ϩ T cells with anti-CD3/IL-2 in the presence of IL-4, IL-10, or TGF-␤. IL-4 markedly enhanced the downregulation of IL-18R␣ expression observed when T cells were cultured with anti-CD3 and IL-2 (Fig. 5, A, B, I, and K). While the addition of IL-10 had no effect (Fig. 5 C), the addition of TGF-␤ augmented IL-18R␣ expression (Fig. 5 D). Although T cells primed in the presence of IL-4 or IL-10 expressed detectable levels of the IL-18R␣, they failed to secrete IFN-␥ in response to IL-18 alone (Fig. 5, F and G). However, these cells were capable of producing significant quantities of IFN-␥ when cultured with both IL-12 and IL-18 (Fig. 5, F-H). Because these studies were performed without concomitant neutralization of IL-12 and/or IFN-␥, it is possible that the inability of IL-4 to completely inhibit IFN-␥ induction by IL-12 and IL-18 is due to endogenous IL-12/IFN-␥.
Because the addition of exogenous IL-4 was capable of downregulating IL-18R␣, we suspected that endogenous IL-4 was partially responsible for the downregulation of IL-18R␣ observed with anti-CD3 stimulation in the absence of any added cytokine (Fig. 5 A). To test this hypothesis, we stimulated T cells from IL-4 Ϫ/Ϫ mice and measured IL-18R␣ expression. When compared with wild-type C57BL/6 mice, IL-4 Ϫ/Ϫ T cells expressed significantly higher levels of IL-18R␣ after anti-CD3 stimulation (Fig.  5, I and J). Levels of IL-18R␣ expression before stimulation were comparable for both C57BL/6 and IL-4 Ϫ/Ϫ mice (data not shown). However, the addition of exogenous IL-4 resulted in a downregulation of IL-18R␣ that was comparable to that seen with wild-type cells (Fig. 5, K and L). These results confirm a critical role for IL-4 in downregulation of IL-18R␣.
Requirement for Stat6 in Regulation of IL-18R␣ Expression/ Function on CD4 ϩ T Cells. Because IL-4 signals mainly via Stat6, we determined if Stat6 signaling was required for IL-4-induced downregulation of IL-18R␣ after T cell activation. After stimulation with anti-CD3 and IL-2, CD4 ϩ T cells from Stat6 Ϫ/Ϫ mice expressed higher levels of IL-18R␣ (⌬MFI ϭ 208) compared with wild-type T cells (⌬MFI ϭ 93; Fig. 6, A and D). This suggests that endogenous Stat6 signaling partially contributes to anti-CD3induced downregulation of IL-18R␣, which is consistent with the results obtained with IL-4 Ϫ/Ϫ mice (Fig. 5, I-K). In contrast to the downregulatory effects of IL-4 seen with wild-type T cells (Fig. 6 B), the addition of IL-4 to cultures of CD4 ϩ T cells from Stat6 Ϫ/Ϫ mice had no effect on IL-18R␣ expression (Fig. 6 E) indicating that Stat6 is required for IL-4-mediated downregulation of IL-18R␣ expression. Interestingly, the ability of IL-12 to augment IL-18R␣ ex- pression was significantly enhanced in the absence of Stat6 (Fig. 6, C and F). To correlate the differences in receptor expression with the ability of IL-18 to induce IFN-␥ production, anti-CD3-primed T cells from both BALB/c and Stat6 Ϫ/Ϫ mice were restimulated with either immobilized anti-CD3 or cytokine and subsequently stained for intracellular IFN-␥. T cells from Stat6 Ϫ/Ϫ mice produced more IFN-␥ in response to anti-CD3 stimulation, as well as the combination of IL-12 and IL-18 (Fig. 6, G-K). Importantly, IL-18-induced IFN-␥ production was significantly enhanced in Stat6 Ϫ/Ϫ mice (13 vs. 7%, Fig. 6 J), which is consistent with the higher levels of IL-18R␣ expression observed on Stat6 Ϫ/Ϫ mice (Fig. 6 D). These results suggest that IL-4 and Stat6 are critical regulators of both IL-18R␣ expression and IL-18 responsiveness.
CD4 ϩ T Cells Primed under Th1 Conditions Upregulate IL-18R␣. Since we found that all naive CD4 ϩ T cells expressed the IL-18R␣, it was of interest to examine the kinetics of regulation of expression during the differentiation of naive CD4 ϩ T cells into Th1 and Th2 cells. For this purpose, we used T cells from 5CC7 transgenic mice on the RAG-2 Ϫ/Ϫ background to minimize the number of previously activated T cells in the starting population. Th1 cells were generated by culturing purified CD4 ϩ T cells with APCs, antigen, IL-12, and anti-IL-4 as described previously (21). After 1 wk in culture, the cells were restimulated with antigen and APCs. Intracellular cytokine staining revealed that 45% of 1-wk (1ϫ) Th1 cells expressed IFN-␥ (Fig. 7 A). No IL-4 could be detected in this population. 1-wk Th1 cells expressed high levels of IL-18R␣ before restimulation (Fig. 7 D), but these levels increased upon stimulation with APCs and antigen (Fig. 7 G). Upon restimulation, Th1 cells secreted large amounts of IFN-␥ after culture with IL-12 and IL-18, but also secreted significant amounts of IFN-␥ when cultured with IL-18 alone (Fig. 7  J). The null cells were generated by priming with peptide, APCs, and IL-2 in the presence of anti-IL-4, anti-IL-12, and anti-IFN-␥ antibodies. Although these cells proliferated vigorously when restimulated with peptide and APCs, they failed to differentiate into Th1 or Th2 cells as judged by the lack of intracellular staining for IFN-␥ or IL-4 ( Fig.  7 B). The level of IL-18R␣ on Th null cells after 1 wk of culture and restimulation was similar to that seen on unstimulated CD4 ϩ T cells (Figs. 7, E and H, and 1 A). Thus, neither IL-12 nor IFN-␥ is required for maintenance of the basal level of IL-18R␣ expression during the 1-wk priming period. These results are consistent with those obtained with anti-CD3-stimulated T cells from IFN-␥ Ϫ/Ϫ (Fig. 4) and IL-12 Ϫ/Ϫ (data not shown) mice. Interestingly, Th null cells failed to produce IFN-␥ when restimulated with IL-12 or IL-18 alone, but did respond to the combination of IL-12 and IL-18 ( Fig. 7 K).
CD4 ϩ T Cells Primed under Th2 Conditions Rapidly Downregulate IL-18R␣ Expression. The results obtained with Th1 cells (Fig. 7) are consistent with the observation that terminally differentiated Th1 cells express high levels of IL-18R␣. To understand the dynamics of IL-18R␣ during Th2 differentiation, TCR transgenic CD4 ϩ T cells were cultured in the presence of antigen, APCs, IL-4, anti-IL-12, and anti-IFN-␥. After 1 wk of priming, 22% of the cells expressed intracellular IL-4, but not IFN-␥, when restimulated with antigen and APCs (Fig. 7 C). However, expression of the IL-18R␣ was markedly downregulated after 1 wk of priming under Th2 conditions (Fig. 7, F and I). More impressively, Th2 cells failed to secrete any detectable IFN-␥ when cultured with IL-12, IL-18, or both IL-12 and IL-18 (Fig. 7 L). Thus, signaling via IL-12 and IL-18 receptors is extinguished even after 1 wk of priming under Th2 conditions.

Discussion
IL-18 has now been well characterized as a costimulatory factor for IL-12-mediated Th1 differentiation. IL-18 may mediate its effects directly by augmentation of IFN-␥ production (6,10,23,24) or by enhancement of the expression of the IL-12R complex, thereby potentiating the effects of IL-12 (25). While many studies have focused attention on the regulation of the IL-12R during Th cell differentiation, fewer have focused on regulation of the IL-18R (24,(26)(27)(28)(29). It has been widely assumed that the IL-18R␣ is expressed only after T cell activation as the IL-18R␣ could readily be detected on fully differentiated murine Th1 cells, but not on Th2 cells, either at the mRNA level or at the cell surface (22)(23)(24)26). We have used a polyclonal antibody prepared against residues 19-346 of the extracellular domain of the IL-18R␣ and were readily able to detect IL-18R␣ on almost all CD4 ϩ and CD8 ϩ T cells. It is highly likely that our ability to detect the IL-18R␣ on naive cells is related to both the specificity of the antibody we have used as well as to our staining protocol.
Although the IL-18R␣ was expressed at relatively uniform levels on the majority of CD4 ϩ T cells, we detected a subpopulation of unstimulated CD4 ϩ T cells that expressed -01-5فfold higher levels of receptor than the majority of the CD4 ϩ population. These cells appeared to be activated/ memory T cells based on their coexpression of other markers (CD62L low , CD44 hi , and data not shown). In contrast to the majority of CD4 ϩ IL-18R␣ int cells, the CD4 ϩ IL-18R␣ hi cells upregulated expression of the IL-18R␣ on culture with IL-12 alone. It is very likely that the effects of IL-12 alone on IL-18R␣ that have been reported by others (23) are secondary to stimulation of this memory T cell population. Indeed, in our hands fully differentiated Th1 clones will also upregulate IL-18R␣ in response to IL-12 and produce IFN-␥ in the absence of TCR ligation. Therefore, it is unlikely that truly naive cells can respond to IL-12 by upregulating the IL-18R␣, especially since these cells do not express the IL-12R␤2 subunit which is required for IL-12 signaling. We have not yet studied the regulation of the IL-18R␣ on CD8 ϩ T cells in response to IL-12 and it also remains possible that the IL-18R␣ is differentially regulated on CD8 ϩ T cells. In the studies of Yoshimoto et al. (23), no effort was made to separate CD4 ϩ cells and CD8 ϩ cells.
Short-term culture of CD4 ϩ T cells in IL-2 alone led to downregulation of IL-18R␣ on the CD4 ϩ IL-18R␣ int population, and this downregulatory effect was further potentiated by stimulation via the TCR. These data are consistent with a previous report showing anti-CD3-triggered downregulation of IL-18R␣ expression (23) and with the observations of Ahn et al. (30) that culture of a Th1 clone with IL-2 alone also led to downregulation of IL-18R␣ expression. In contrast to the findings of Yoshimoto et al. (23), we found that culture of CD4 ϩ T cells with the combination of anti-CD3 and IL-12 led to a pronounced increase in IL-18R␣ expression on the CD4 ϩ IL-18R␣ int population. A possible difference between the results presented here and those of others is that in our 4-d culture system with anti-CD3 and cytokines, T cells were cultured with immobilized anti-CD3 for only the first 2 d to reduce cell death as the result of repeated stimulation via the TCR. Culture of CD4 ϩ T cells with anti-CD3 and IL-18 failed to induce upregulation of the IL-18R␣. However, IL-18 could synergize with IL-12 to induce IL-18R␣ upregulation in the presence of anti-CD3. Collectively, these results are most consistent with a model in which IL-12 dictates IL-18 responsiveness in CD4 ϩ T cells. Further evidence of this is supported by the observation that the ability of IL-12 to upregulate IL-18R␣ expression is abrogated in Stat4 Ϫ/Ϫ mice (data not shown).
The capacity of IL-12 to induce upregulation of the IL-18R␣ was completely IFN-␥ dependent, as T cells from IFN-␥ Ϫ/Ϫ mice did not upregulate their IL-18R␣ when stimulated with anti-CD3 and IL-12. Although reconstitution of these cultures with IFN-␥ restored IL-18R␣ expression, IFN-␥ itself did not enhance IL-18R␣ expression in the absence of IL-12. Thus, IFN-␥ increases T cell responsiveness to IL-12 mostly likely via its effects on induction/maintenance of expression of the IL-12R␤2 chain.
The detection of IL-18R␣ on resting CD4 ϩ T cells prompted us to examine the factors responsible for the absence of the IL-18R␣ on Th2 clones. We have demonstrated that IL-4 rapidly downregulates the expression of the IL-18R␣ during short-term stimulation of naive CD4 ϩ T cells with anti-CD3. When CD4 ϩ T cells were primed under Th2 conditions, marked downregulation was seen after 1 wk of culture and after 3 wk of culture under Th2 conditions, there was no detectable IL-18R␣ expression (data not shown). There was no observable downregulation of IL-18R␣ when resting T cells were cultured with IL-4 (data not shown) in the absence of TCR stimulation, suggesting that TCR-mediated signals are also required. The effect of IL-4 on IL-18R␣ expression was even more dra- Figure 7. Regulation of IL-18R␣ expression during Th1/Th2 differentiation. CD4 ϩ T cells from 5CC7 transgenic mice were primed with irradiated APCs and cytochrome C under Th1, Th null, or Th2 conditions. After 1 wk in culture, T cells were rechallenged for 6 h with PMA and ionomycin and then stained for intracellular IFN-␥ and IL-4 to confirm polarization of T cells into Th1 (A), Th null (B), and Th-2 (C) phenotypes. Alternatively, T cells were rechallenged for 2 d with APCs and antigen, but in the absence of cytokines. Cells were analyzed for IL-18R␣ expression both before (D-F) and after (G-I) rechallenge. Also, Th1 (J), Th null (K), or Th2 (L) cells were restimulated with the indicated cytokine and levels of IFN-␥ determined by ELISA. matic when T cells from IL-4 Ϫ/Ϫ mice were used. IL-4 Ϫ/Ϫ T cells expressed higher levels of IL-18R␣ after the 4-d culture when compared with wild-type cells; however, the addition of exogenous IL-4 resulted in downregulation of IL-18R␣ to levels similar to wild-type. This is consistent with the notion that endogenous IL-4 contributes to IL-18R␣ downregulation during anti-CD3 stimulation. Further support for the role of IL-4 in negative regulation of IL-18R␣ is that Stat6 Ϫ/Ϫ T cells, which are impaired in their ability to respond to IL-4, also express higher levels of IL-18R␣ after T cell activation. In addition, the ability of IL-12 to upregulate IL-18R␣ is enhanced in Stat6 Ϫ/Ϫ T cells. Surprisingly, the addition of TGF-␤ to either anti-CD3 or antigen-primed cultures led to an increase in IL-18R␣ expression. TGF-␤ has been shown by others to inhibit IL-4 production (31), and we hypothesize that the inhibition of IL-4 production by TGF-␤ is responsible for the observed increase in IL-18R␣ expression.
In addition to examining the cytokine requirements for expression of the IL-18R␣ during Th1/Th2 differentiation, we have also examined their effects on the induction of both IL-12 and IL-18 responsiveness. Restimulation of CD4 ϩ T cells that had been cultured for 4 d with anti-CD3, IL-2, and the combination of IL-12 and IL-18, but not either cytokine alone, resulted in production of large amounts of IFN-␥. More importantly, when CD4 ϩ T cells were cultured for 4 d with anti-CD3 and IL-12, they produced significant amounts of IFN-␥ when restimulated with either IL-12 or IL-18 alone. Identical results were observed with 1-wk Th1 cells. The capacity to respond to IL-18 alone correlated with enhanced level of cell surface IL-18R␣ expression. Although CD4 ϩ T cells cultured with anti-CD3 and IL-4 for 4 d retained their capacity to respond to the combination of IL-12 and IL-18, 1-wk Th2 cells were completely nonresponsive. This is probably due to the absence of endogenous IL-12 and IFN-␥ in Th2priming experiments. Similar results have been reported by others (7,32). The ability of IL-4 to regulate IL-18induced IFN-␥ production was also evident in T cells from Stat6 Ϫ/Ϫ mice, which secreted significantly more IFN-␥ in response to IL-18/IL-12 plus IL-18 when compared with controls. This enhanced production of IFN-␥ correlated with increased levels of IL-18R␣ expression. Although IFN-␥ production in response to IL-18 appeared to correlate with expression of the IL-18R␣, we cannot exclude the possibility that increased expression/induction of IL-18R␤ accounted, in part, for enhanced IFN-␥ production in response to IL-18. Very little information is available concerning the regulation of IL-18R␤ expression (20).
Intriguingly, 1-wk Th null cells still responded vigorously to the combination of IL-12 and IL-18 by producing significant amounts of IFN-␥. These in vitro findings raise a number of questions about the possible role of IL-12 and IL-18 in inducing IFN-␥ production during the course of an inflammatory response in vivo. As IL-12 and IL-18 may be produced by different cell types under different conditions, one possibility is that T cells primed in an IL-12 rich environment will subsequently be able to respond by pro-ducing IFN-␥ in response to either cytokine alone in the absence of reactivation by their TCR. Although the in vivo equivalent of our Th null cell population has not been directly demonstrated, immunization of mice with autoantigens under certain conditions results in a population of primed T cells that only produce IL-2 and not Th1/Th2 cytokines (33)(34). It is possible that when these cells are exposed to IL-12 and IL-18 in a paracrine fashion during the course of an infectious insult they would complete their differentiation into pathogenic Th1 cells.
Apart from the known effects of IL-18 in synergizing with IL-12 to drive Th1 responses, recent reports have shown that IL-18 can induce Th2-type responses (i.e., IgE, IL-4, IL-13) (12-15). One interpretation of these studies is that IL-18 is a potent inducer of IL-4 and IL-13 production by basophils and mast cells, or possibly CD4 ϩ T cells. Indeed, induction of IL-4 production by IL-18 in NK T cells has recently been reported (35), which is consistent with our observation that unstimulated CD4 ϩ NKT cells express high levels of IL-18R␣ similar to NK cells (data not shown). Such a systemic production of IL-4 may facilitate the priming of antigen-specific CD4 ϩ Th2 cells. Our observation that IL-4 leads to downregulation of the IL-18R␣ on CD4 ϩ T cells is very compatible with such a model, as the absence of the IL-18R␣ on the antigenresponsive T cells would further prevent the potential costimulatory effects of IL-18 on IL-12-mediated Th1 differentiation. Since this IL-4-mediated downregulation of IL-18R␣ was only observed when T cells were triggered through the TCR, bystander effects of IL-4 on resting cells that express the IL-18R␣ would not occur. Ultimately, the major factor that determines whether IL-18 potentiates Th1-or Th2-type immune responses is likely to be the balance between IL-12 and IL-4 in the microenvironment during T cell priming. The presence of IL-12, as well as the expression of IL-12 receptor components, would allow IL-18 to synergize with IL-12 in the development of Th1 development. Conversely, the presence of specialized cell types capable of making IL-4 in response to IL-18 (i.e. mast cells) would favor IL-18 as a potentiator of Th2 responses.