We have demonstrated that TCR-induced IL-21R expression is regulated at the level of transcription in human T cells and depends on the synthesis of new proteins. Interestingly, the induction pattern is biphasic in primary T cells. This pattern may be due to the involvement of different factors that regulate early/late induction. In the early induction phase, IL-21R mRNA induction requires the synthesis of new or activation of preexisting transcription factors. We hypothesize that the late induction of IL21R
gene expression may result from cytokines produced by activated T cells. For example, IL-21 is known to up-regulate expression of its own receptor (57
In examining the molecular mechanism underlying the transcriptional regulation of the IL21R
gene, we found that Sp1 binds to a GC-rich motif in the proximal promoter and mediates IL-21R induction in activated human T cells. Sp1 is known to activate many vital genes, including genes involved in cell growth and development (5
), and homozygous deletion of Sp1 in mice causes severe lethal embryonic malformation (30
). Classically, Sp1 was viewed as a constitutive transcription factor that regulates the basal expression of many cellular genes (7
). However, it has now been found to be involved in tissue-specific gene expression and in the control of transcription following different stimuli (3
In the immune system, Sp1 has been reported to control the expression of genes that mediate important cellular functions. For example, Sp1 regulates the expression of genes encoding IL-2Rβ (26
), TCR Vα (19
), and FasL (54
) and plays a critical role in regulating the division of T lymphocytes after costimulation with anti-CD2 and anti-CD28 (22
). We now show that Sp1 plays an essential role in regulating TCR-induced IL-21R expression. Mutation of the Sp1 binding sites in the IL-21R proximal promoter region diminished PI-induced reporter activity. Strikingly, Sp1 expression was significantly increased in primary T cells after TCR stimulation. The TCR-induced augmentation of Sp1 transcriptional activity correlated with induction of IL-21R mRNA, and transfection of Sp1 siRNAs into primary human T cells significantly decreased TCR-induced IL-21R mRNA expression. Unexpectedly, however, Sp1 expression is only slightly increased 2 h after TCR activation, while IL-21R mRNA levels were highly induced at this time point. We therefore hypothesized that posttranslational modification of Sp1 might play a critical role for IL-21R gene expression. Depending on cell type and stimuli, phosphorylation of Sp1 has been reported to either increase (41
) or decrease (1
) Sp1 transcriptional activity. We thus examined the effect of the phosphatase inhibitors calyculin A and okadaic acid, which inhibit PP1 and PP2A activity, and found that they decreased IL-21R expression. Moreover, the phosphatase inhibitors increased the phosphorylation of Sp1 and correspondingly decreased Sp1 DNA-binding activity for the IL-21R promoter. These results suggest that posttranslational modification of Sp1 is critical for TCR-induced IL-21R expression.
Sp1 has been reported to exert its transcriptional activation with the help of other comodulators or cofactors, such as NF-κB (20
), STAT proteins (6
), Smad family factors (33
), Egr family factors (26
), NFAT family proteins (54
), or other members of the Sp family (9
). Complex C2, as detected in EMSA, appears to represent Sp3, since anti-Sp3 diminished the formation of C2. However, the DNA-binding activity of complex C2 was only slightly induced by PI activation, in contrast to Sp1, whose binding activity was markedly induced. In addition, siSp3 had little effect on TCR-induced IL-21R expression, and the cotransfection of siSp1 and siSp3 was not more potent than siSp1 transfection alone. Thus, unlike Sp1, Sp3 plays only a minor role in regulating IL-21R repression. So far, we do not yet have definitive data that support factors other than Sp1 as critically contributing to the regulation of IL-21R. Since PI activation is associated with protein kinase C activation and calcium/calcineurin pathway, it is conceivable that other factors, such as AP-1, NF-κB, or NFAT (10
), might be involved in IL-21R regulation, an area for future investigation.
Taken together, our results reveal that Sp1 is indeed a critical regulator of IL21R gene expression, a finding with implications as to how the expression of this critical receptor can be controlled. Moreover, the observation that Sp1 is potently induced and dephosphorylated in response to anti-CD3 plus anti-CD28 has much broader implications for the regulation of Sp1-dependent genes in antigen-activated T cells. Further studies are needed to clarify how TCR activation induces the dephosphorylation of Sp1 and to identify the relevant target residues. Such studies will not only further clarify the basis for IL-21R regulation but also help us to understand how Sp1-regulated genes in general are broadly controlled in the immune system.