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
Trang 1kinase Tec
Liang Yu1,2, Oscar E Simonson1, Abdalla J Mohamed1 and C I Edvard Smith1
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, Ca2+ 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.
Trang 2neutrophils [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
Trang 3we 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 inhibitranscrip-tion of the NF-jB
A
B
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.
Trang 4signaling 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 database version 7.0 [33] and
p-match 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 l M ) 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.
Trang 5to 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.
Trang 6constructs 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 signalaffect-ing 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 n M ) 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 l M ) or Bortezomib (20 n M ) 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.
Trang 7directly 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 signalaffect-ing (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 propro-moter–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.
Trang 8the 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 hepatolympho-cytes, 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¢-TCACTAGATCTGA 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
Trang 9M2 (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 monitordur-ing 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 TRANSFAC database
version 7.0 (http://www.biobase-international.com) [33] and
p-match 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
Acknowledgements
This work was supported by the Swedish Cancer Fund, the Wallenberg Foundation, the Swedish Science Council, the Swedish Research Council, the Swedish Foundation for Strategic Research, the Swedish Hemophilia Society and Stockholm County Council (research grant ALF)
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