NF-jB regulates the transcription of protein tyrosine kinase Tec Liang Yu 1,2 , Oscar E. Simonson 1 , Abdalla J. Mohamed 1 and C. I. Edvard Smith 1 1 Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 2 Department of Hematology, Huaian NO. 1 hospital, Nanjing Medical University, Huaian, Jiangsu, China Introduction The tyrosine kinase expressed in hepatocellular carci- noma (Tec) is a non-receptor tyrosine kinase belonging to the Tec family of protein tyrosine kinases (PTK). It was originally cloned from a mouse liver cDNA library [1], but was later shown to be particularly important for hematopoietic cell development [2–5]. Tec family PTKs (TFKs) comprise five members: Tec, Btk (Bruton’s tyrosine kinase), Itk, Bmx and Txk. These kinases are involved in a wide range of signaling path- ways that control mitogen-activated protein kinase (MAPK) activation, Ca 2+ influx, actin reorganization, transcriptional regulation, cell survival and cellular transformation [6–11]. Initially, it was thought that Tec expression was tis- sue-specific, as it could only be detected in hepatocytes and liver tumor cells. However, it was later shown that Tec is also expressed in hematopoietic cells, such as B and T lymphocytes, myeloid lineage cells and human Keywords Bortezomib; hydrodynamic transfection; NF-jB; Tec; transcription Correspondence L. Yu, Department of Hematology, Huaian No. 1 Hospital, Nanjing Medical University, Huaian 223300, Jiangsu, China Fax: +86 517 84907078 Tel: +86 517 84952303 E-mail: liang.yu@ki.se C. I. E. Smith, Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden Fax: +46 8 58583650 Tel: +46 8 58583651 E-mail: edvard.smith@ki.se (Received 10 June 2009, revised 5 September 2009, accepted 16 September 2009) doi:10.1111/j.1742-4658.2009.07385.x The tyrosine kinase expressed in hepatocellular carcinoma (Tec) is a non- receptor protein tyrosine kinase (PTK) that is expressed in hematopoietic cells, such as B and T lymphocytes, myeloid lineage cells and neutrophils. Mutations in the human Btk gene cause X-linked agammaglobulinemia (XLA), but the corresponding mutation in mice results in a much milder defect. However, the combined inactivation of Btk and Tec genes in mice cause a severe phenotype resembling XLA. Tec is involved in the regula- tion of both B and T lymphocytes, fine-tuning of TCR ⁄ BCR signaling, and also activation of the nuclear factor of activated T cells. Previous work has shown that the transcription factors Sp1 and PU.1 can bind and regulate the Tec promoter. In this study, we demonstrate that NF-jB is an essential transcription factor for optimal expression of the Tec gene, and identify a unique functionally active NF-jB binding site in its promoter. The NF-jB subunit p65 ⁄ RelA directly induced transcriptional activity of the Tec pro- moter. Moreover, we also found that proteasome inhibitors, including Bortezomib, repress Tec transcription through inactivation of the NF-jB signaling pathway. This study, together with our previous findings on the transcriptional regulation of Btk (Bruton’s tyrosine kinase) by proteasome inhibitors, provides important insight into the molecular mechanism(s) underlying the role of NF-jB in Tec family kinase signaling and lympho- cyte development. Abbreviations Btk, Bruton’s tyrosine kinase; I-jB, inhibitor of jB; PEST, penicillin/streptomycin; PTK, protein tyrosine kinase; Tec, tyrosine kinase expressed in hepatocellular carcinoma; TFKs, Tec family PTKs; XLA, X-linked agammaglobulinemia. 6714 FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS neutrophils [3,4,12–16]. Moreover, Tec and other mem- bers of this group show a high degree of conservation across species [11,17,18]. Previous studies have shown that Tec plays impor- tant roles in regulating the function of B and T lym- phocytes, as well as of other types of cells. Kitanaka et al.[12] showed that Tec is activated in both mature and immature B-lymphoid cells, and is therefore impli- cated in B-cell development and activation. In primary T cells, downregulation of Tec expression by an anti- sense strategy causes reduction in production of the interleukin IL-2 in response to TCR stimulation [19]. In contrast, overexpression of Tec in the T-cell line Jurkat synergizes with the action of phorbol myristate acetate to induce activation of the nuclear factor of activated T cells, whereas Itk overexpression has no effect [20]. These data suggest that Tec is important for optimal TCR signaling. Isolated Tec deficiency in experimental animals does not manifest itself in the form of disease; however, knockout mice lacking both Btk and Tec (Btk - ⁄ - ⁄ Tec - ⁄ - ) show a severe phenotype resembling X-linked agammaglobulinemia (XLA) [2]. In addition, Btk-deficient cell lines can be functionally reconstituted using Tec [21,22]. Tec activation and phosphorylation also play critical roles in the process of IL-1 and IL-8 secretion, and in the generation of chemotactic activity in supernatants from stimu- lated neutrophils [23]. More recently, it was reported that Tec is involved in Fcc receptor-induced signal- ing and phagocytosis [17,18], as well as regulation of osteoclast differentiation [17,18]. Moreover, Tec has also been shown to be involved in the intracellular signaling of a number of cytokines, such as IL-3, IL-6, erythropoietin and granulocyte colony-stimulating factor [4,24–26]. Although molecular cloning of the mouse Tec pro- moter was reported more than 10 years ago [27,28], additional regulatory factors have not been identified, with the exception of the transcription factors SP1 and PU.1. Recently, we identified two functionally active NF-jB sites in the promoter region of Btk, and demonstrated that proteasome inhibitors repress Btk transcription through inhibition of the NF-jB signaling pathway [29]. Interestingly, we also found that proteasome inhibitors reduce the steady-state levels of Tec. NF-jB is known to be important for both innate and adaptive immunity, and is essential for T- and B-lymphocyte-mediated antigen-specific defense. The NF-jB ⁄ Rel family of proteins includes NF-jB1 (p50), NF-jB2 (p52), RelA (p65), c-Rel and RelB, which form functional homo- or heterodimer complexes [30,31]. Following activation, NF-jB binds to regulatory elements in the promoter region of target genes. More- over, NF-jB is crucial for induction of host defense genes during acute pathogenic threats in insects as well as vertebrates [31,32]. In this study, we aimed to investigate the role of NF-jB signaling in the regulation of Tec expression. We identified a conserved, functionally active NF-jB site in the Tec promoter, and found that the NF-jB subunit p65 ⁄ RelA potently induced the Tec promoter in B and T lymphocytes, as well as in the liver cell lines HepG2 and Huh7. Furthermore, we show that proteasome inhibitors reduce the expression of both human and mouse Tec following inactivation of the NF-jB signaling pathway. Collectively, these findings shed light on the molecular mechanisms underlying NF-jB regulation of gene expression of TFKs, and the subsequent effect on lymphocyte development, expres- sion and the immune response. Results Characterization of the mouse Tec promoter Molecular cloning of the mouse Tec promoter has been described previously [27,28]. To create a reporter construct under the control of the Tec promoter, geno- mic DNA from the mouse cell line A20 was amplified by PCR and cloned into the promoterless pGL3-basic vector. In total, three reporter constructs correspond- ing to various lengths of the mouse Tec promoter were generated (Fig. 1A). To determine the activity of the Tec promoter, the reporter constructs were introduced into the B-cell line A20. As shown in Fig. 1C, the shortest construct, Tec-420 (promoter region from position )384 relative to the transcription initiation site), displayed the highest luciferase activity. This result is consistent with a previous report showing that a Tec promoter construct starting from position )364 had the strongest transcriptional activity [27]. In con- trast, we found that transcription from the construct containing the promoter region starting from position )894 (Tec-920 construct) had only 60% activity com- pared with the Tec-420 construct. In contrast, the pre- vious study [27] showed that a promoter construct starting from )1006 had almost the same activity as one starting from )364. This discrepancy may be due to the different cell lines used for reporter expression. We repeated our experiment in the Jurkat T-cell line as well as in heterologous cell lines (COS7 and 293T), and obtained the same results (data not shown). To further determine the in vivo expression of the Tec promoter, we employed the hydrodynamic infusion method, a robust in vivo gene delivery technique that L. Yu et al. NF-jB regulates protein tyrosine kinase Tec FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS 6715 we have used previously to functionally characterize promoter activities in live mice [29]. Using this proce- dure, we introduced the shortest construct (Tec-420) into the liver of mice, and monitored the luciferase- derived signal by bioluminescence imaging. Our results show that this reporter construct was highly active in liver, and that its expression lasted for a long period of time (more than 1 month) (Fig. 1D and data not shown). Thus, the Tec promoter is active in both hematopoietic and liver cells. Proteasome inhibitors suppress the Tec promoter Previously, we demonstrated that proteasome inhibi- tors can decrease the expression of Tec. In the present study, we also showed that proteasome inhibitors can decrease Tec expression in mouse primary B and T cells (data not shown). However, we did not investi- gate whether proteasome inhibitors affect Tec expres- sion at the transcriptional or post-transcriptional levels [29]. To resolve this issue, we transfected the Tec-420 construct into the B-cell line A20, and treated the cells 36 h later with proteasome inhibitors (MG132 or Bortezomib) or with the specific NF-jB inhibitor Bay 11-7085. Both proteasome and NF-jB inhibitors sig- nificantly decreased the transcriptional activity of the Tec promoter (Fig. 2). We replicated this experiment in the T-cell line Jurkat and in two liver cell lines, HepG2 and Huh7 (Fig. 5C, left part, and data not shown). The proteasome and NF-jB inhibitors repress transcription from the Tec-920 and Tec-1800 promo- ters in all tested cells (data not shown). These results indicate that proteasome and NF-jB inhibitors use the same mechanism for regulating expression of the Tec tyrosine kinase. NF-jB binds directly to the Tec promoter and induces Tec transcription The above results show that both proteasome and NF-jB inhibitors can suppress the transcriptional activity of the Tec promoter. In our previous work, we showed that proteasome inhibitors repress transcrip- tion of the TFK Btk through inhibition of the NF-jB A B CD Fig. 1. Characterization of Tec promoter–luciferase reporter constructs. (A) Schematic representation showing the structure of mouse Tec promoter–luciferase reporter constructs Tec-420, Tec-920 and Tec-1800; two putative NF-jB binding sites are indicated, and the known PU.1 and SP1 binding sites are also shown. (B) Schematic representation showing the potential NF-jB binding sites in human and mouse Tec promoters. (C) Transcriptional activity of the Tec promoter–luciferase reporter constructs. Tec-420, Tec-920 or Tec-1800 (5 lg) were transfected into A20 mouse B lymphocytes, and cells were lysed 48 h later and subjected to luciferase activity analysis; relative levels of luciferase activity are shown. Data are representative of three independent experiments. (D) Expression of Tec promoter–luciferase reporter constructs in the liver of living mice. NMRI mice were injected with 10 lg Tec-420 by the hydrodynamic procedure. At day 2 post-injection, the luciferase-derived signal was measured by the IVIS imaging system as described in Experimental procedures. NF-jB regulates protein tyrosine kinase Tec L. Yu et al. 6716 FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS signaling pathway [29]. We were interested to deter- mine out whether a similar mechanism also operates in the case of Tec. To address this question, we first per- formed a computational scan of the promoter region, approximately 2 kb upstream of the Tec transcription start site, for transcription factor binding sites using the public TRANSFAC Ò database version 7.0 [33] and p-matchÔ software [34]. The scan results indicated two putative NF-jB binding sites in the mouse Tec promoter (Fig. 1A). To further determine the func- tional relevance of these sites, we used a chromatin immunoprecipitation (ChIP) assay. As Fig. 3A shows, the NF-jB subunit p65 ⁄ RelA readily associated with the Tec promoter. Having shown that NF-jB binds to the Tec promoter, we then determined whether this site is functionally utilized. Accordingly, we co-transfected a p65-expressing plasmid (pcDNA1-p65) with the Tec promoter–luciferase reporter constructs into A20 cells. Figure 3B shows that the promoter activities of all three constructs increase following overexpression of p65. Furthermore, when the Tec-420 construct was co-transfected with increasing amounts of p65 into A20 cells, there was a dose-dependent increase in promoter activity. In addition, when similar amounts of p65 and Tec-420 were introduced into the cells, luciferase activity increased 2.3-fold compared to base- line, and when twice the amount of p65 was used, the luciferase activity increased 3.5-fold over baseline (Fig. 3C). This result indicates that NF-jB is recruited Fig. 2. Proteasome and ⁄ or NF-jB inhibitors regulate Tec promoter transcription. The Tec-420 luciferase reporter construct was intro- duced into A20 cells by electroporation, and cells were treated 32 h later without or with Bortezomib (20 n M), MG132 (10 lM)or Bay 11-7085 (5 l M) for another 16 h. Luciferase activity was measured, and the relative levels of luciferase activity are shown. Data are representative of three independent experiments. A B C D Fig. 3. NF-jB binds directly to the Tec promoter and induces Tec transcription. (A) The chromatin of A20 cells was cross-linked, sheared and immunoprecipitated with the indicated antibodies. Input and immunoprecipitated DNAs were purified and used as templates in PCR with primers specific for the Tec promoter (region from )406 to )127 that contains the putative NF-jB binding site). Primers for PU.1 were used as a positive control (PU.1 has been reported to bind the same region); rabbit normal IgG was used as a negative control. (B–D) Tec reporter constructs and the corresponding mutants were co-transfected with pcDNA1-p65 into A20 cells as indicated; 48 h later, cells were lysed and subjected to luciferase activity analysis. The relative levels of luciferase activity are shown. Data are representative of three independent experiments. L. Yu et al. NF-jB regulates protein tyrosine kinase Tec FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS 6717 to the regulatory region of the Tec promoter and induces expression of Tec. A single NF-jB site is active in the mouse Tec promoter As there are two putative NF-jB binding sites in the Tec promoter (Fig. 1A), we were interested to know whether both sites could functionally be utilized. To investigate whether one or both sites are active, site- directed mutagenesis was used to generate mutant versions of the reporter constructs. To this end, all constructs and their corresponding mutants were co-transfected with or without the p65 expression plas- mid into A20 cells. As shown in Fig. 1A, the shortest construct Tec-420 harbors a single putative NF-jB binding site (NF- jB binding site a), whereas Tec-920 contains two putative NF-jB binding sites, NF-jB binding site a and NF-jB binding site b. In compari- sion with the wild-type, when NF-jB binding site a was mutated (Tec-420M), p65 expression was not able to induce promoter activity (Fig. 3D), suggesting that this NF-jB site is functionally active. The activity of the Tec-920 reporter construct also increased following expression of the NF-jB subunit p65. Mutating the second NF-jB binding site (site b) (construct Tec-920M2) did not affect the transcriptional induc- tion by p65. In sharp contrast, when NF-jB binding site a was mutated alone (Tec-920M1) or together with NF-jB binding site b (Tec-920M1 + M2), transcrip- tional induction by the NF-jB subunit of p65 was abolished (Fig. 3D). Accordingly, when NF-jB bind- ing site a was mutated, the baseline Tec promoter transcription activity decreased. Thus Tec-420M had 67% of the activity of Tec-420, and Tec-920M1 had 65% of the activity of Tec-920 (Fig. 3D). These findings indicate that NF-jB is an essential transcrip- tion factor for expression of the mouse Tec gene. Taken together, our results clearly show that there is a functionally active NF-jB binding site in the Tec promoter and that NF-jB subunit p65 induces its transcriptional activity in hematopoietic cells. In vivo analysis of the mouse Tec promoter In our previous work, we found that a Btk promoter– reporter construct could be successfully expressed in the liver of young mice [29]. As Tec is normally expressed in liver cells, we decided to assess the activity of the Tec promoter in live mice. We introduced reporter con- structs containing the Tec-420 and Tec-420M promot- ers into the liver of adult NMRI mice by hydrodynamic transfection, and monitored the luciferase-derived signal by bioluminescence imaging. As expected, the Tec promoter constructs were highly active in liver tissue of these mice. Interestingly, wild-type and mutant constructs showed similar expression levels in the first week of the assay (Fig. 4). However, 7 days later, the expression levels of the mutant construct had declined sharply, and no luciferase-derived signal could be detected at day 14. In contrast, expression of the wild-type Tec promoter construct was shown to be very stable after day 7 and persisted for more than 1 month (Fig. 4 and data not shown). This result indicates that NF-jB is not critical during the initial expression of Tec in the liver. However, NF-jB may be indispensable for long-term expression, as mutation of the NF-jB binding site leads to a significant reduction in the steady-state levels of the reporter gene. Expression of Tec requires NF- jB signaling Under normal physiological conditions, the p65 ⁄ p50 heterodimer is sequestered in an inactive form in the cytoplasm by the inhibitor of jB (I-jB). Following stim- ulation, I-jB is phosphorylated by I-jB kinase, leading to its ubiquitination and subsequent degradation by the proteasome pathway [35–38]. Consequently, free NF-jB rapidly enters the nucleus and activates target gene expression [39,40]. We have demonstrated that protea- some inhibitors reduce Tec transcription (Fig. 2), and that the NF-jB subunit p65 induces the Tec promoter (Figs 3 and 5). To investigate the effect of proteasome inhibitors on Tec transcription, Tec-420 and Tec-420M Fig. 4. Expression of Tec promoter–luciferase reporter constructs in vivo. NMRI mice were injected with 10 lg Tec-420 or Tec-420M using the hydrodynamic procedure. The luciferase-derived signal was measured at days 1, 3, 7 and 14 post-injection using the IVIS imaging system as described in Experimental procedures. Data are representative of two independent experiments. NF-jB regulates protein tyrosine kinase Tec L. Yu et al. 6718 FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS constructs were transfected into A20 cells, and cells were treated with proteasome inhibitors 36 h later. As shown in Fig. 5A, proteasome inhibitors did not affect the transcriptional activity when NF-jB binding site a was mutated. Similar results were obtained when Tec-920 and mutants thereof were tested (data not shown). Co-transfection of the Tec-420 construct with p65 into A20 cells cultured in the presence or absence of MG132 or Bay 11-7085 significantly increased Tec-420 activity (Fig. 5B). In contrast, when proteasome and NF-jB inhibitors were added to the cell culture, the luciferase activity decreased to the original level (Fig. 5B). Similar results were obtained when liver cell line HepG2 was tested (Fig. 5C). These findings further confirm that the proteasome inhibitor-induced block of Tec promoter transcription is an NF-jB-dependent signaling phenom- enon, indicating that NF-jB plays an important role in controlling Tec family kinase transcription. NF-jB regulates the human Tec promoter As the NF-jB signaling pathway regulates the mouse Tec promoter, we wished to determine whether this phenomenon is conserved in other species. We there- fore investigated the human Tec promoter. As shown in Fig. 1, the human Tec promoter also contains a putative NF-jB site, corresponding to the same position in the mouse Tec promoter. The human Tec promoter was cloned into the luciferase reporter construct, generating the plasmid pGL3-Hu-Tec. This reporter construct was transfected together with pcDNA1-p65 plasmid into the human B-cell line Nam- alwa. We found that NF-jB ⁄ p65 modestly induces the human Tec promoter in these cells (1.7-fold), and that the proteasome inhibitor MG132 significantly reduced the transcriptional activity (Fig. 6). Similar results were obtained in the cell lines A20, U937 and HEK293T (data not shown). Collectively, our results show that NF-jB can bind to both human and mouse Tec pro- moters and induce their transcriptional activity, and proteasome and NF-jB inhibitors instead compromise their activity, suggesting that this regulation has been conserved during evolution. Discussion In this study, we identified NF-jB as an essential tran- scription factor for optimal expression of the Tec gene, and found that the NF-jB subunit p65 ⁄ RelA can A B C Fig. 5. Proteasome inhibitors suppress Tec transcription by affect- ing the NF-jB signaling pathway. (A) Tec-420 or Tec-420M were introduced into A20 cells by electroporation, and cells were treated 32 h later without or with MG132 (10 l M) or Bortezomib (20 nM) for another 16 h as indicated. Luciferase activity was measured and the relative levels of luciferase activity are shown. (B,C) The Tec-420 construct was co-transfected without or with pcDNA1-p65 into A20 cells (B) or HepG2 cells (C). Cells were treated 32 h later with MG132 (10 l M), Bay 11-7085 (5 lM) or Bortezomib (20 nM) for another 16 h as indicated. Luciferase activity was measured and the relative levels of luciferase activity are shown. Data are repre- sentative of three independent experiments. L. Yu et al. NF-jB regulates protein tyrosine kinase Tec FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS 6719 directly activate the Tec promoter. Moreover, we also found that proteasome inhibitors, including the only one currently approved for clinical use, Bortezomib, reduce Tec transcription. In our previous work, we found that proteasome inhibitors reduce the steady- state levels of Tec [29]. In the present study, we dem- onstrated that these inhibitors repress transcription of the Tec gene. NF-jB is important for both innate and adaptive immunity, and is thus essential for B- and T-lympho- cyte-mediated antigen-specific defense. In addition, NF-jB is critical for the survival of developing B lym- phocytes and the maturation of spleen B cells [41–43]. Abnormal activation of NF-jB has been shown to be a hallmark of the development and progression of lym- phoid malignant diseases [44–47]. Tec is expressed in both mature and immature B-lymphoid cells, and has important roles in regulating the function of B and T lymphocytes during development and activation [12]. In this study, we found that Tec expression is trans- criptionally regulated by the NF-jB signaling pathway. Moreover, Btk, another TFK that is known to be critical for B-cell proliferation, differentiation and acti- vation [11,48], is also transcriptionally regulated by NF-jB [29]. Taken together, our findings shed light on the underlying mechanism(s) of NF-jB as a regulator of lymphocyte survival, proliferation, differentiation and disease development. Although Tec has many important physiological functions, the transcriptional regulatory mechanisms of Tec have not been fully elucidated. Here, we identified a functionally active NF-jB site in the regulatory region of the Tec promoter (Fig. 1A). The NF-jB subunit p65 ⁄ RelA induced Tec transcription, and the baseline transcriptional activity decreased to two-thirds of the original level when the NF-jB binding site was mutated. Moreover, the stimulatory activity of p65 on the Tec promoter was completely abrogated (Fig. 3). Interestingly, two putative NF-jB binding sites were found in the mouse genome using in silico analysis (Fig. 1A). However, only the evolutionarily conserved site, NF-jB binding site a, was shown to be functionally active, and p65 can be recruited to bind to the promoters and induce transcription of both human and mouse Tec (Figs 3 and 6). Protein ubiquitination is critical in regulating funda- mental cellular processes, such as cell-cycle regulation, endocytosis, antigen presentation and apoptosis [49,50]. The ubiquitin proteasome pathway controls key cellular and biological processes and therefore plays crucial roles in health and disease, and there is increasing evidence that this pathway is also involved in regulating gene transcription [29]. In this study, we found that the ubiquitin proteasome pathway has important roles in regulating Tec gene transcription, and proteasome inhibitors dramatically decrease the transcriptional activity of the Tec promoter by affect- ing NF-jB signaling (Figs 2 and 5). Our results, together with those of others, suggest that, under nor- mal conditions, NF-jB, Sp1 and PU.1 are essential transcription factors that maintain the baseline tran- scriptional activity of the Tec promoter [27]. Following mutation of NF-jB binding site, the baseline transcrip- tion activity of Tec promoter was decreased (Figs 3C and 5A). When proteasome inhibitors are used, I-jB degradation is blocked, and the accumulated cytoplas- mic I-jB blocks nuclear translocation of NF-jB, thereby abrogating transcription of the Tec promoter (Figs 2 and 5). Tec is differentially expressed in cells and tissues, and its expression level is approximately 15–17-fold lower than that of Itk and Btk in T cells and B cells, respectively. In B cells, Tec expression is approximately 5-fold higher than in T cells, but Tec is upregulated in activated T cells and in Th1 ⁄ Th2 effector cells [51]. Although Tec expression levels are known to vary among different cell types, the underlying mechanism of such variation is not known. Our results clearly demonstrate dose dependence of the induction of Tec promoter transcription by NF-jB (Fig. 3C). This may partly, but not exclusively, explain why Tec is differen- tially expressed in cells, for example, NF-jB signaling is constitutively active in A20 cells, and we found that Fig. 6. The NF-jB signaling pathway regulates human Tec pro- moter transcription. The human Tec promoter–luciferase reporter construct pGL3-Hu-Tec was co-transfected without or with pcDNA1-p65 into Namalwa cells by electroporation, and cells were treated 32 h later without or with MG132 (10 l M) for another 16 h. Luciferase activity was measured and the relative levels of lucifer- ase activity are shown. Data are representative of three indepen- dent experiments. NF-jB regulates protein tyrosine kinase Tec L. Yu et al. 6720 FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS the Tec expression level is higher in A20 cells than in other tested cells (data not shown). Finally, using the hydrodynamic transfection method, we successfully introduced the Tec promoter– luciferase reporter construct (Tec-420) into the liver of mice. Tec expression was very high in the liver of mice, and the luciferase-derived signal could still be detected 1 month later (Figs 1D and 4). This result indicates that the Tec promoter could be an interesting tool for exogenous gene expression in vivo. It appears that NF- jB is not important for the initial expression of Tec in the liver, but is critical for sustained expression (Fig. 4). The underlying mechanism is not known and requires further investigation. In the liver, Tec has been shown to be an early response gene that enhances hepatocyte proliferation and liver regeneration [52]. Of note is the fact that the Tec promoter behaves in a similar way in cell lines of both hepatic and hemato- poietic origin. This may indicate that the altered reac- tivity of liver cells in vivo reflects the proliferative state of these cells, as only a minor proportion of cells in the liver are dividing at any one time. Thus, it may well be that the Tec promoter in resting hematopoietic cells is also not subject to the same NF-jB regulation as in activated, proliferating cells. The NF-jB signaling pathway plays critical roles in liver physiology and disease. Knockout mice that are deficient in RelA, but not other NF-jB family members, die prematurely due to extensive hepatocyte apoptosis [53]. In contrast, targeted depletion of some of the com- ponents in this pathway did not result in pronounced sensitivity of hepatocytes to tumor necrosis factor a-or lipopolysaccharide-induced apoptosis [54,55]. Thus, in the liver, NF-jB signaling predominantly protects hepatocytes from tumor necrosis factor a-induced apop- tosis. Moreover, the same pathway has been shown to be important during liver pathogenesis [56]. It is inter- esting that NF-jB plays a crucial role in both lympho- cytes and hepatocytes, as both cell types also express Tec, which itself is regulated by NF-jB. In summary, we identified a conserved and function- ally active NF-jB site in the Tec promoter, and found that the NF-jB subunit p65 ⁄ RelA induces Tec promoter transcription activity. Furthermore, we also found that proteasome inhibitors can reduce Tec transcription via the NF-jB signaling pathway. These findings provide further insight into the transcriptional regulation of the TFKs and the role of NF-jB signaling in lymphocyte development and the immune response. Moreover, the increased use of drugs influencing NF-jB signaling requires a through understanding of genes regulated by this signaling pathway in order to define their mechanism of action. Experimental procedures Reagents Anti-Tec antibody was purchased from Santa Cruz Biotech- nology (Santa Cruz, CA, USA); protease and phosphatase inhibitors have been described previously [57]. MG132 and Bay 11-7085 were purchased from Sigma (St Louis, MO, USA). Bortezomib was obtained from Millennium Pharma- ceuticals (Cambridge, MA, USA). Polyclonal anti-p65, anti-PU.1 and rabbit normal IgG were purchased from Santa Cruz Biotechnology. Cell culture and transfections HepG2 and Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% v ⁄ v heat- inactivated fetal bovine serum and 1% penicillin ⁄ strepto- mycin (PEST) (Invitrogen, Carlsbad, CA), and cells were transfected with FuGENE 6 reagent (Roche Applied Sci- ence, Indianapolis, IN, USA) according to the manufac- turer’s instructions. HEK293T, COS7 and A20 cells were cultured and transfected as previously described [58]. Jurkat and Namalwa cells were maintained in RPMI-1640 medium supplemented with 10% v ⁄ v heat-inactivated fetal bovine serum and 1% PEST, and cells were transfected by electro- poration in a 0.4 cm gap cuvette at 250 V and 960 micro- farads using a Bio-Rad (Hercules, CA, USA) gene pulser. Plasmid constructs and luciferase assay The mouse Tec promoter–luciferase reporter constructs Tec-1800 ()1776 ⁄ +45), Tec-920 ()894 ⁄ +45), and Tec-420 ()387 ⁄ +45) were amplified by PCR using mouse genomic DNA, and verified by sequencing. The PCR fragment was digested using BglII and HindIII and subcloned into the pGL3-Basic vector (Promega, Madison, WI, USA). The PCR primers for Tec-1800 were 5¢-TCACTAG ATCTGA ATGAGAGGCAGGAGAGAA-3¢ (forward) and 5¢-AGTG AAAGCTTAAGACAGAGCGACGTCCAAA-3¢ (reverse). Those for Tec-920 were 5¢-TCACTAGATCTTGTCTCTCT CCTTCTGAGAG-3¢ (forward) and 5¢-AGTGAAAGCTT AAGACAGAGCGACGTCCAAA-3¢ (reverse). The prim- ers for Tec-420 were 5¢-GTTACAGATCTAGCACAGCAT CATCCGGTTT-3¢ (forward) and 5¢-AGTGAAAGCTTAA GACAGAGCGACGTCCAAA-3¢ (reverse). Site-directed mutagenesis was used to generate mutant versions of mouse Tec promoter–luciferase reporter constructs. Tec-420M and Tec-920M1 (NF-jB binding site a mutations) were created by changing the NF-jB binding sequence GGATGGGA AGTCCGG to GGATTTTGGGTCCGG, and Tec-920M2 (NF-jB binding site b mutation) was created by changing the NF-jB binding sequence AAATGGGCTTGCCTT to AAATAAATCTGCCTT. Finally, the Tec-920M1 + M2 mutant was created by combining Tec-920 mutants M1 and L. Yu et al. NF-jB regulates protein tyrosine kinase Tec FEBS Journal 276 (2009) 6714–6724 ª 2009 The Authors Journal compilation ª 2009 FEBS 6721 M2 (binding sites a and b). All constructs were verified by DNA sequencing. The human Tec promoter–luciferase reporter construct pGL3-Hu-Tec ()427 ⁄ +67) was amplified by PCR using human genomic DNA, and verified by sequencing. The PCR fragment was digested with BglII and HindIII and subcloned into the pGL3-Basic vector. The PCR primers for pGL3-Hu-Tec were 5¢-AGTATAGATCTGTGC GGTTCCTAATTCCGACAG-3¢ (forward) and 5¢-ATGTC AAGCTTCCTTACCTGGCTGAAGCGC-3¢ (reverse). Plas- mid pcDNA1-p65 (full-length human p65 ⁄ RelA expressed from a cytomegalovirus promoter) was obtained from Rune Toftga ˚ rd (Karolinska Institutet, Stockholm, Sweden). Luci- ferase activity was measured as previously described [29]. Immunoprecipitation and immunoblotting Cells were routinely analyzed 48 h post-transfection. Immu- noprecipitation and immunoblotting were performed essen- tially as described previously [58]. Hydrodynamic transfection Hydrodynamic transfections of plasmids in Ringer solution were carried out as previously described [29,59,60]. Briefly, 8% v ⁄ w Ringer solution containing 10 lg Tec promoter– luciferase reporter construct was introduced by tail vein injection over a period of 5 s to inbred NMRI mice. Live, anesthetized mice were imaged for 10 s to 5 min using an intensified CCD camera (IVIS imaging system, Xenogen, Hopkinton, MA, USA). The animals where anesthetized with isofluran (4% induction, 2.5% maintenance dose) dur- ing the injection and monitoring of gene expression. Ethical permission All animal research was approved by the Local Committee for Animal Ethics in Stockholm, Sweden, and was performed in accordance with this ethical permission and European Community directive 86 ⁄ 609 ⁄ EEC. All animal experiments were designed to minimize the suffering and pain of the animals. Bioinformatic tools used for identification of transcription factor binding sites in the Tec promoter In silico analysis was performed on the Tec promoter region. Sequence corresponding to 2 kb upstream of the Tec transcription start site was analyzed. The computa- tional scan for transcription factor binding sites was per- formed using the public TRANSFACÒ database version 7.0 (http://www.biobase-international.com) [33] and p-matchÔ software [34]. A matrix (TFP60pm) was chosen, and three scans were performed using alternative parameter settings to minimize the false negative or positive rates with regard to the Tec promoter sequence. Chromatin immunoprecipitation assay (ChIP) The ChIP assay was performed essentially as reported pre- viously [29,61]. The primers for the NF-jB binding sequence were 5¢-CCATTCTTCTATCACCCCAG-3¢ (for- ward) and 5¢-TCCTTCCGCGATTTCAAAGC-3¢ (reverse). The values obtained from immunoprecipitated samples were normalized to those of input samples. 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Pelanda R, Reth M, Rajewsky K & Schmidt-Supprian M (2006) Canonical NF-jB activity, dispensable for B cell development, replaces BAFF-receptor signals and promotes B cell proliferation upon activation Immunity 24, 729–739 44 Davis RE, Brown KD, Siebenlist U & Staudt LM (2001) Constitutive nuclear factor jB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells J Exp . China Introduction The tyrosine kinase expressed in hepatocellular carci- noma (Tec) is a non-receptor tyrosine kinase belonging to the Tec family of protein tyrosine kinases. and NF-jB inhibitors use the same mechanism for regulating expression of the Tec tyrosine kinase. NF-jB binds directly to the Tec promoter and induces Tec