From the
Liver Group, Division of Infection, Inflammation and Repair, University of Southampton, Southampton SO16 6YD, United Kingdom and the ¶Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737
Received for publication, October 29, 2003 , and in revised form, April 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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By far the most well understood NF-B signal transduction pathway is that which leads to the degradation of the best characterized I
B protein, I
B
(3). Following the stimulation of cells by activating agents such as proinflammatory cytokines, mitogens, and viral proteins, I
B
is rapidly phosphorylated at two serine residues (Ser-32 and Ser-36) by the recently discovered I
B kinase complex (3, 8). Phosphorylated I
B
is then a substrate for ubiquitin ligases that catalyze the addition of ubiquitin groups to I
B
; this modification then targets I
B
for degradation by the 26 S proteasome (3). Degradation of I
B
releases active NF-
B; however, this activation is only short lived as NF-
B stimulates transcription of the I
B
gene leading to a rapid replenishment of the inhibitor (1013). The newly synthesized I
B
enters the nucleus where it binds to NF-
B dimers leading to their dissociation from DNA and return to the cytoplasm (14). The end result is a replenishment of the cytoplasmic pool of inducible NF-
B and diminution of NF-
B-dependent transcription back to its basal state.
The classical transient NF-B activation pathway operates in many cell types to generate a rapid but short lived burst of NF-
B activity that enables the cell to mount an appropriate response to changes in its microenvironment. However, it is also apparent that levels of basal NF-
B activity can differ greatly between different cell types. These differences presumably reflect the individual cellular requirements for NF-
B-dependent gene expression. For example, although many mammalian cells express low levels of active NF-
B, other cell types such as B cells, thymocytes, macrophages, neurons of the cortex and hippocampus, and cultured proliferating smooth muscle cells have been found to express relatively high basal levels of NF-
B (1522). In addition, elevation of constitutive NF-
B activity has been reported for several different types of tumor cell, in aged animals and during the terminal differentiation of monocyte-derived dendritic cells (2226). The mechanisms that regulate basal NF-
B activity in cells are relatively poorly understood. One mechanism has been documented in lipopolysaccharide-stimulated cells that undergo a persistent activation of NF-
B (27). In these cells a hypophosphorylated form of I
B
is expressed which binds Rel dimers in such a way that nuclear localization and DNA binding are not impeded. As interactions between I
B
or I
B
with NF-
B are mutually exclusive, hypophosphorylated I
B
functions as a chaperone protecting the active transcription factor from inhibition by I
B
(27). Other mechanisms for long term elevation of NF-
B activity have been documented in Reed-Steenberg cells of Hodgkin's lymphoma. These cells have been reported to exhibit reduced expression of I
B
, which either results from amino acid mutations that reduce stability of the I
B
protein or from higher constitutive I
B kinase activity (2830). Despite these examples, little is understood about the signaling mechanisms that regulate basal NF-
B activity in the majority of differentiated mammalian cells.
We have reported previously (31, 32) that activation of hepatic stellate cells (HSC),1 a pivotal event in hepatic fibrosis, is associated with a persistent elevation of basal NF-B activity. Activated HSC are the major source of the excess extracellular matrix proteins that ultimately distort the normal architecture and function of the liver in individuals suffering from a wide range of chronic liver diseases (33). In addition, activated HSC express an impressive array of proinflammatory and profibrogenic factors, many of which are regulated at the level of gene transcription by NF-
B(e.g. tumor necrosis factor-
, IL-6, IL-8, MCP-1, and ICAM1) (34, 35). NF-
B has also been shown to be protective against cytokine-induced HSC apoptosis (36). Given the strong pro-inflammatory milieu of the activated HSC, its elevated expression of active NF-
B is therefore likely to be an important regulator of cell survival and in turn the persistence and progression of liver fibrosis. We noticed that the re-programming of basal NF-
B activity during culture-induced activation of primary rat HSC (a widely accepted model for in vivo HSC activation) was associated with dramatically reduced expression of cytoplasmic and nuclear I
B
(31). We reasoned that this diminution of I
B
during HSC activation may be one way in which the cell re-programs its basal NF-
B activity. Moreover, we were interested in the possibility that culture-induced activation of primary HSC may provide a model system in which to discover the mechanisms by which cells program their constitutive levels of active NF-
B.
In this study we provide evidence that HSC activation is associated with induced expression of the ubiquitously expressed transcriptional repressor RBP-J (hereafter termed CBF1), which is able to bind to a subset of NF-B sites that overlap with consensus CBF1-binding sites (3740). We have mapped an overlapping NF-
B/CBF1-binding site (
B2 site) in the human I
B
promoter and have shown that overexpression of CBF1 resulted in a profound repression of I
B
gene transcription and protein expression. We demonstrate that this effect requires direct interaction of CBF1 with the
B2 site and results in an elevation of NF-
B activity. We also show that the intracellular domain of Notch1 can de-repress I
B
gene transcription and protein expression via a CBF1-dependent mechanism that operates synergistically with the transcriptional co-activator p300. These findings provide novel I
B kinase-independent mechanisms by which cells regulate NF-
B activity.
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MATERIALS AND METHODS |
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Plasmid DNAAll plasmid DNA was prepared using a commercial DNA extraction and isolation kit (Maxiprep, Qiagen). IB
promoter function was studied using the luciferase reporter vector pI
B
wt-Luc vector containing nucleotides 332 to +35 of the human gene promoter (provided by Professor Ron Hay (St. Andrews, UK)). The control Renilla luciferase reporter vector driven by a thymidine kinase promoter (pRLTK) was purchased from Promega (Southampton, UK). CBF1 expression vector pJH282 (provided by Dr Diane Hayward, Baltimore, MD) has been described previously (44). Expression vector for CBF1 lacking DNA binding activity (pR218H) was obtained from Professor Tasuku Honjo (Kyoto, Japan) (45). Expression vectors 4wtCBF1-Luc (pJH26A) and 4xmutCBF1-Luc (pJH28A) luciferase reporter vectors, pBos-ZEDN1 (Notch1 IC), pBos-CDN1 (Notch1 IC containing the ankyrin repeat and pest domain), pBos-CDCN1T (Notch1 IC containing the ankyrin repeat domain only) have been described elsewhere (48, 52). Expression vectors pCMV-HA-p300 and pCMV were obtained from Issay Kitabayashi (Tokyo, Japan). Expression vector pCMV-HA-p53 was obtained from Dr. Jeremy Blades (Southampton, UK).
Chromatin Immunoprecipitation (ChIP) AssayChIP assay was carried out on 2 x 106 quiescent or activated rat hepatic stellate cells from the same preparation using immunoprecipitation (ChIP) assay kit as per the manufacturer's instructions (Upstate Biotechnology, Inc.). Antibodies used for immunoprecipitation were polyclonal goat anti-CBF1, rabbit anti-p50 (both from Santa Cruz Biotechnology), and rabbit anti-acetylated histone H4 (Upstate Biotechnology, Inc.). PCR amplification of IB
promoter was carried out using specific oligonucleotide primers selected within the promoter region of the gene. Sense primer sequence was 5'-cgc taa gag gaa cag cct ag-3', and antisense primer sequence was 5'- cag ctg gtc gaa aca tgg c-3'.
Co-culture of LTK/SN3T9 with Transfected HSCRat HSC were transfected with pJH26A or pJH28A and pRLTK for 24 h and then trypsinized and co-cultured on either on a confluent monolayer of LTK (parental L-cells)/SN3T9 (Jagged1 expressing L-cells) or placed in transwell which was itself placed in wells with LTK or SN3T9 cells for a further 24 h. Cells were then harvested, and luciferase assays were performed using a dual luciferase kit (Promega) according to the manufacturer's instructions. Expression of firefly luciferase was normalized for differences in transfection efficiency by measurement of the activity of pRLTK.
EMSANF-B binding was determined by EMSA as described previously (31, 32), using a 32P-end-labeled double-stranded oligonucleotide probe containing either wild type or mutated NF-
B sites or NF-
B sites as found within the human I
B
promoter sequence. Nuclear extracts were prepared as described previously, and their protein content was determined using the Bradford DC assay kit (Bio-Rad) (31, 32). Wild type NF-
B sense oligonucleotide was 5'-tga ggg gac ttt ccc agg 3'; the p50/p65 mutant sense oligonucleotide was 5'-tga ggc gac ttt ccc agg 3', and the CBF1 mutant sense oligonucleotide was 5'-tga ggg gac ttc ccg agg 3'. Mutated bases are underlined and in boldface type. NF-
B sites within human I
B
promoter were used as probes, namely
B-like 1 (5'-ccc aga gaa atc ccc agc cag-3'),
B1 (5'-ggc ttg gaa att ccc cga gcc-3'),
B-like 2 (5'-cgc agg gag ttt ctc cga tga-3'),
B3 (5'-gtc gga aag act ttc cag cca-3'),
B2 (5'-atc gtg gga aac ccc agg gaa-3'), and
B2 mutant which no longer binds CBF1 (5'-atc gtc ggg aac ccc agg gaa-3', mutated bases are underlined and in boldface type). For supershift assays, reactions were incubated in the presence of 2 µg of anti-p50, anti-p65, anti-c-Rel, anti-CBF1, anti-JunD, or anti-JunB antiserum (Santa Cruz Biotechnology). EMSA and supershift reaction mixtures were resolved by electrophoresis on an 8% non-denaturing polyacrylamide gel (37:5:1).
FACS Analysis100 µl of 1 x 106/ml quiescent rat HSC were incubated with 15 µl of rabbit anti-Notch1 polyclonal antibody, or rabbit anti Notch2 antibody, or irrelevant rabbit polyclonal antibody (all at 200 µg/µl) (Santa Cruz Biotechnology). Cells were incubated with primary antibody for 30 min at 4 °C and washed with phosphate-buffered saline/bovine serum albumin/azide. Cells were then labeled with 10 µl of sheep anti-rabbit fluorescein isothiocyanate conjugate (Sigma) for a further 30 min and washed as before. Analysis by flow cytometry was carried out on FACSCalibur (BD Biosciences). Activated HSC were dissociated from the flask by 10 min of incubation in citric saline at 37 °C and processed for analysis following the protocol described above.
Total RNA Isolation and cDNA SynthesisTotal RNA was isolated from 1 x 107 quiescent (day 1) and activated (day 7) rat HSC using the Total RNA purification kit (Qiagen, UK) following the manufacturer's instructions. First strand cDNA was generated using 1 µg of total RNA, 1 µl of random hexamer primer (p(dN)6), and RNase-free water (Qiagen, UK), heated at 70 °C for 5 min, and then placed on ice. RNasin (RNase inhibitor), 100 units of Moloney murine leukemia virus-reverse transcriptase, 1x Moloney murine leukemia virus-reverse transcriptase buffer, and 0.4 mM dNTPs were added, and the mix was incubated at 42 °C for 1 h.
Reverse Transcriptase (RT)-PCRPCR amplification of Notch1, Notch2, Notch3, Notch4, and -actin cDNAs was carried out using specific oligonucleotide primers selected within the coding regions of the genes. CBF1 amplification was carried out using oligonucleotides designed to cross-react with both mouse and human CBF1 coding regions due to a lack of published rat CBF1 sequence. Notch1 primers used were 5'-cct ggg tgg atg gga aca aac-3' (sense) and 5'-gaa aag ccg ccg aga tag tca-3' (antisense) designed to produce a 1277-bp product; Notch2 primers were 5'-ggg cta cac tgg gaa aaa ctg-3' (sense) and 5'-ctg ggg gac aac gac aaa tga-3' (antisense) designed to produce an 848-bp product; Notch3 primers were 5'-cgg gat gtg gat gaa tgt ctg-3' (sense) and 5'-agg ggc tgc gga agg ggt ctc-3' (antisense) designed to produce a 1482-bp product; Notch4 primers were 5'-gac cgt gtg ggc tct ttc tcc-3' (sense) and 5'-ggg ctc tgt gtg cct gac ctt-3' (antisense) designed to produce an 829-bp product; CBF1 primers were 5'-cct cag caa gcg gat aaa-3' (sense) and 5'-gac atg gag tgg cct gaa-3' (antisense) designed to produce a predicted 473-bp product, and
-actin primers were 5'-aga ggg aaa tcg tgc gtg aca-3' (sense) and 5'-aca tct gct gga agg tgg aca-3' (antisense) designed to produce a 453-bp product. PCRs were composed of 1 µl of cDNA template, 100 ng each of sense and antisense oligonucleotide primers, 2.5 µl of optimized TaqPCR buffer (Promega), 0.4 mM dNTP mixture, and 2 units of Taq polymerase in a total reaction volume of 25 µl. Following an initial 5-min incubation at 94 °C, PCRs were performed using a 1-min annealing step (at 59.3 °C for Notch1, 58.3 °C for Notch 2, 60.5 °C for Notch 3, 59.8 °C for Notch 4, 52 °C for CBF1, and 57.2 °C for
-actin), followed by a 2-min elongation step at 72.0 °C and a 45-s denaturation step at 94 °C. A total number of 35 PCR cycles were carried out for amplification of all cDNAs (unless otherwise stated in the figure legend), followed by a final elongation reaction for 10 min at 72.0 °C. PCR products were separated by electrophoresis at 50 V for 90 min through a 1% agarose gel and were detected by ethidium bromide staining. Expected sizes of specific PCR products were verified by reference to a 1-kb DNA ladder.
SDS-PAGE and ImmunoblottingWhole cell extracts were prepared, and protein concentration of samples was determined using Bradford DC assay kit (Bio-Rad). 30 or 50 µg of whole cell extracts from rHSC or transfected COS cells, respectively, were then fractionated by electrophoresis through a 9% SDS-polyacrylamide gel. Gels were run at a 100 V for 1.5 h prior to transfer onto nitrocellulose. Following blockade of nonspecific protein binding, nitrocellulose blots were incubated for 1 h with primary antibodies diluted in TBS/Tween 20 (0.075%) containing 3% Marvel. Mouse monoclonal horseradish peroxidase-conjugated antibody recognizing FLAG tag of pJH282 was used as 1:20,000 dilution for direct detection of the tagged protein (Sigma). Rat monoclonal antibody directed against the Myc tag of R218H was used at a final concentration of 1 µg/ml. J59 antibody directed against Jagged1 protein was used as described previously (69). Rabbit polyclonal antibody recognizing IB
was used at a final concentration of 1 µg/ml. Goat polyclonal antibody recognizing endogenous CBF1 was used at a dilution of 1:8000. Mouse monoclonal antibody recognizing
-actin was used at a dilution of 1:1000 (all from Santa Cruz Biotechnology). Blots were then washed three times in TBS/Tween 20 prior to incubation for 1 h with sheep anti-rabbit horseradish peroxidase antibody for I
B
, rabbit anti-goat horseradish peroxidase conjugate antibody for CBF1, goat anti-mouse horseradish peroxidase conjugate antibody for
-actin, and rabbit anti-rat horseradish peroxidase conjugate antibody for Myc-tagged CBF1 mutant expressed from R218H expression vector (all antibodies were used at 1:2000 dilution in TBS/Tween 20 (0.075%) containing 3% Marvel). After extensive washing in TBS/Tween 20, the blots were processed to distilled water for detection of antigen using the ECL system (Amersham Biosciences).
Site-directed Mutagenesis of IB
I
B
PromoterThe mutagenesis was carried out using two-step recombinant PCR. The first reaction was set up using 100 ng of
B2
CBF1F sense primer (5'-ctt tcc ctg ggg ttc ccg acg atc g-3', mutated bases are in boldface and underlined) and 100 ng of I
B
CBF1prR antisense primer (5'-ttc gag ctc ggt acc cgg gga tc-3'), 500 ng of pI
B
wt-Luc template, 2.5 µl of optimized Pfu polymerase buffer (Promega), 0.4 mM dNTP mixture, and 2 units of Pfu polymerase in a total reaction volume of 25 µl. PCR was carried with an initial 5-min incubation at 94 °C. This was followed by 40 cycles of a 1-min annealing step at 58 °C, a 2-min elongation step at 72 °C, and a 45-s denaturation step at 94 °C. After 40 cycles a final elongation reaction was carried out for 10 min at 72 °C. The 90-bp PCR product was separated by electrophoresis and purified, and 5 µl of the eluted product was then used in second step PCR, along with 500 ng of pI
B
wt-Luc template and 100 ng of I
B
CBF1prF2 sense primer (5'-caa gct tgg acg gcg gca cgg a-3'). The PCR program was set up as described for the first step reaction with two annealing steps, first at 62 °C for 45 s, followed by another 45 s at 48 °C. PCR was carried out for 40 cycles followed by a final extension at 72 °C for 10 min. The PCR product was than digested with HindIII and SalI in a 37 °C water bath and separated on 1% agarose gel, excised, purified, and ligated into pGL3 Basic. The presence of the required mutation was confirmed by sequencing.
Transfections and Reporter Gene AssaysHSC and COS cells were transfected by the non-liposomal Effectene protocol (Qiagen) according to the manufacturer's instructions. Luciferase assays were performed using a dual luciferase kit (Promega) according to the manufacturer's instructions. pJH26A (4xCBF1wt), pJH28A (4xCBF1mut) and pIB
wt promoter-driven expression of firefly luciferase was normalized for differences in transfection efficiency by measurement of the activity of a co-transfected Renilla vector pRLTK.
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RESULTS |
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After 710 days in culture HSC adopt a proliferative myofibroblast-like phenotype that closely resembles the phenotype of in vivo activated HSC (33). By using this culture model we have reported previously (31) that HSC activation is associated with a persistent elevation in NF-B DNA binding activities inclusive of an uncharacterized high mobility DNA-protein complex. In order to define further the regulation of NF-
B activity in HSC, we sought to identify the high mobility complex. Sequence analysis of the oligonucleotide probe used for EMSA studies in our previous work revealed the presence of an overlapping CBF1-binding site (Fig. 1A). Nucleotide substitutions expected to perturb binding of either p50:p65 or CBF1 were introduced into the NF-
B EMSA probe to test the idea that the high mobility complex was due to CBF1 binding. A point mutation predicted to block p50:p65 binding resulted in loss of the lower mobility complex but had no effect on the higher mobility complex. By contrast, a double point mutation that from previous studies (40) was predicted to disrupt CBF1 binding caused a loss of the higher mobility complex and did not affect formation of the lower mobility complex. These data indicated that CBF1 is responsible for the higher mobility complex and were confirmed by supershift/antibody interference EMSA analysis which showed strong immunoreactivity of the high mobility complex with anti-CBF1 (Fig. 1B). We next determined whether HSC activation is associated with changes in CBF1 expression that would explain induction of the CBF1 protein-DNA complex. Western blot analysis revealed that freshly isolated and early cultured rat HSC lack detectable CBF1 expression. However, HSC cultured for 7 and 10 days expressed high levels of a protein species that was recognized by the CBF1 antibody and that was of a similar size (57 kDa) to CBF1 expressed in COS1 cells transfected with the CBF1 expression vector pJH282 (Fig. 2A). RT-PCR for detection of rat CBF1 mRNA expression was carried out on RNA isolated from various time points of culture activation of HSC using primers based on the murine CBF1 cDNA sequence predicted to generate a product of 473 bp. At 25 PCR cycles a single product of 473 bp that was confirmed by DNA sequencing to correspond to a partial CBF1 cDNA fragment was detectable in all samples with slightly elevated levels in the 10-day HSC track (Fig. 2B). At 30 cycles similar levels of CBF1 transcript were detected across the entire time course. These data suggest that CBF1 expression is apparently regulated during HSC activation by a post-transcriptional mechanism.
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CBF1 Interacts with the IB
Promoter, Represses Transcription, and Elevates NF-
B ActivityWe have reported previously that the protein-DNA complex now identified as containing the transcriptional repressor CBF1 is induced during HSC activation concomitantly with diminution of I
B
expression and induction of NF-
B DNA binding activity (31). To determine whether CBF1 interacts with the I
B
gene promoter, we carried out ChIP assays using anti-CBF1, anti-p50, and PCR primers shown previously (42) to amplify specifically the I
B
gene promoter. As shown in Fig. 3A, we were able to detect binding of both CBF1 and p50 to the I
B
promoter in activated HSC. By comparison a much lower level of the p50-I
B
promoter interaction was detected in quiescent HSC, which is consistent with the low levels of p50:p65 DNA binding activity we have reported previously (31) in quiescent HSC. However, by contrast a CBF1-I
B
promoter interaction was not detected in quiescent HSC, which most probably reflects the lack of detectable CBF1 protein expression in these cells. A ChIP assay was also carried out for comparison of the level of acetylated histone H4 at the I
B
promoter in quiescent and activated HSC (Fig. 3B). Once again we observed a strong induction of association of CBF1 with the promoter upon HSC activation, and this was accompanied by a significant and reproducible diminution in the amount of acetylated H4 detected at the promoter. These data indicate that HSC activation is accompanied by the recruitment of CBF1 and its associated histone deacetylase (HDAC) containing transcriptional co-repressor complexes to the I
B
promoter.
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It has been estimated that up to 12.5% of NF-B-binding sites that have the GGAAA half-site may overlap with CBF1 sites (40). The human I
B
promoter (Fig. 4A) carries three consensus NF-
B-binding sites (
B1 site located at 63 to 53,
B3 site at 225 to 216, and
B2 site at 319 to 310) and two non-consensus NF-
B-like-binding sites (located at 34 to 24 and at 159 to 150) (13, 41, 43). Sequence analysis of the human I
B
promoter indicated the existence of a perfect consensus CBF1-binding site at 321 to 315 which overlaps with the
B2 site. To determine whether CBF1 can specifically interact with this site, we performed EMSA on each of the three
B and two
B-like sites. As shown in Fig. 4A, a single low mobility complex was assembled with the two
B-like sites and the
B1 and
B3 sites. Supershift analysis confirmed that this complex was composed of p50:p65 heterodimers (data not shown). Fig. 4A also shows that the
B2 site forms the p50-p65 complex, but in addition assembles a higher mobility complex that was observed when using nuclear extracts from both Jurkat and COS1 cell lines. CBF1 binding at the
B2 site was confirmed using nuclear extracts from activated HSC; moreover, we also showed that mutation of nucleotides predicted to disrupt CBF1 binding at the
B2 site resulted in a loss of the high mobility complex (Fig. 4B). From these data we concluded that CBF1 binds in a sequence-specific manner to the CBF1 site that overlaps with the 5' end of the
B2 site. We therefore predicted that interaction of CBF1 with the
B2 site should lead to repression of I
B
gene transcription. To test this idea activated HSC were transfected with a wild type I
B
-luciferase promoter or a mutant promoter carrying nucleotide substitutions known to prevent interaction of CBF1 with the
B2 site (as determined in the EMSA in Fig. 4B). The mutated promoter was found to be
8-fold more active than the wild type promoter (Fig. 4C), indicative of a loss of transcriptional repression by CBF1. To confirm a role for CBF1, activated HSC were co-transfected with I
B
-luciferase promoter constructs and an expression vector for CBF1 (pJH282) (44). Fig. 4D shows that cells co-transfected with pJH282 displayed a 10-fold reduction in I
B
promoter activity relative to cells co-transfected with an empty vector. By contrast, cells transfected with an I
B
promoter-luciferase reporter carrying nucleotide substitutions that disrupt CBF1 DNA binding expressed equal promoter activities in the presence and absence of CBF1 overexpression. Furthermore, we were able to show that a mutant CBF1 protein (RBP-J
R218H), which contains a single amino acid mutation that prevents the protein from binding to DNA, was unable to repress I
B
promoter activity when expressed in HSC (45). To determine the ability of CBF1 to repress the expression of endogenous I
B
, we carried out transfection experiments in COS1 cells in which it is possible to achieve high efficiency gene transfer by transfection (up to 80% using Effectene). Transfection of pJH282 caused a dose-dependent repression of I
B
protein expression in COS1 cells (Fig. 5A). Western blot analysis of
-actin confirmed equivalent protein loading across the samples and showed the lack of a general repressive effect of overexpressing CBF1 on COS1 protein expression. As a further control we demonstrated that overexpression of RBP-J
R218H at similar levels to those achieved for wild type CBF1 had no effect on I
B
protein expression (Fig. 5B). As CBF1 can repress I
B
protein, we investigated the effects of diminished I
B
levels on endogenous NF-
B DNA binding activity. EMSA analysis of NF-
B on COS1 cells transfected with CBF1 revealed as expected an increase in the high mobility CBF1-DNA complex; however, in addition we also observed induction of a lower mobility complex (Fig. 5C). Supershift studies indicated that this complex was reactive with antisera recognizing Rel family proteins. Incubation of pre-formed complexes with antisera recognizing p50, p65, and c-Rel subunits generated weak supershift complexes (data not shown); however, incubation with a combination of all three antisera resulted in complete supershift of the lower mobility complex but did not supershift the CBF1-DNA complex (Fig. 5D). We therefore conclude that the lower mobility complex contains NF-
B dimers and that attenuation of CBF1 expression can alter basal NF-
B DNA binding activity.
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Notch Induces IB
Gene Transcription via Interaction with CBF1 and the
B2 SiteFollowing interaction of Notch with a member of the DSL (Delta, Serrate/Jagged, Lag) family of Notch ligands, the intracellular domain of Notch is released and can combine with CBF1 to activate transcription of target genes (46, 47). Analysis of Notch gene family expression was carried out in rat HSC. RT-PCR analysis (Fig. 6A) showed that quiescent HSC express Notch1, -2, and -3 but lack detectable expression of Notch4. Similar analysis of Notch expression in culture-activated rat HSC revealed a similar level of Notch1 and Notch2 mRNA expression as that found in quiescent cells. However, HSC activation was associated with a loss of Notch3 mRNA. Cell surface expression of Notch1 and Notch2 was confirmed by FACS analysis of quiescent and activated rat HSC (Fig. 6B).
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In addition to the RT-PCR and FACS studies, we were able to show that HSC express physiologically functional levels of cell surface Notch proteins by use of a co-culture assay (48). Activated HSC were transfected with synthetic CBF1 reporter constructs that drive transcription from either 4 tandem wild type or mutated (and functionally inactive) CBF1-binding sites upstream from a minimal promoter. The transfected cells were then detached and re-plated onto monolayer cultures of control L cells (cLTK) or L cells stably transfected with a Jagged1 expression vector (SN3T9 cells) that by Western blotting were shown to express high levels of Jagged1 protein (Fig. 6C). For HSC cultured on control L cells, there was no significant difference in activity of the wild type and mutant CBF1 reporters. By contrast, for HSC cultured on SN3T9 cells the wild type CBF1 reporter had a 10-fold higher level of activity than the mutant reporter (Fig. 6D). A similar set of experiments was carried out in which the L cell monolayers were physically separated from transfected HSC by means of a transwell co-culture system. In this transwell system we observed no differences in the activity of wild type and mutant CBF1 reporter activities in both parental L cell and SN3T9 co-cultures. Hence, the stimulatory effect of Jagged1-expressing SN3T9 cells is dependent on direct interaction with HSC and does not involve a soluble factor. These data demonstrate the physiological relevance of studying Notch-mediated transcriptional regulation in HSC. However, a more simplified system was adopted in order to determine whether activation of Notch signaling can stimulate IB
gene transcription.
Several published studies have demonstrated that transfection of expression constructs for the intracellular domain of Notch1 (hereafter referred to as NICD) can be used to mimic the downstream events in the Notch signaling pathway (4951). The ability of an NICD expression construct (ZEDN1) to stimulate the CBF1 reporter by 3.5-fold in transfected activated rat HSC, while having no effect on activity of mutant CBF1 reporter, indicated that this approach is valid for studying downstream Notch signaling events in HSC (Fig. 7A). Transfection of activated HSC with the NICD expression vector enhanced IB
promoter-luciferase activity by up to 5-fold, by contrast activity of the
B2 site mutant promoter construct that lacks CBF1 binding activity was unaffected by overexpression of NICD (Fig. 7B). The mutant CBF1 protein RBP-J
R218H can interact with NICD and function as a dominant negative inhibitor of CBF1/NICD-dependent activation of transcription (45). As shown in Fig. 7C, expression of RBP-J
R218H blocked NICD-induced activation of I
B
promoter activity in HSC. Interaction of NICD with CBF1 requires a fully intact RAM domain, which is composed of two parts known as domains 1a and 1b (52). It has been reported that NICD can also influence gene transcription and cell function by a CBF1-independent route. To establish that the activation of I
B
gene transcription by NICD was due to a CBF1-dependent pathway, we tested the ability of truncated NICD proteins to stimulate I
B
promoter activity. CDN1 is an N-terminal truncation of NICD that lacks most of the RAM domain, whereas CDCN1T is an N- and C-terminal truncation that only leaves the ANK repeat domain (52). Both of these truncation proteins have been shown previously (52) to inhibit the differentiation of C2C12 myoblasts and as such are biologically active proteins. Neither CDN1 nor CDCN1T was able to activate the I
B
promoter in activated HSC confirming that NICD activates I
B
gene transcription via a CBF1-dependent pathway (Fig. 7D). To determine whether NICD can influence endogenous I
B
protein expression, COS1 cells were transfected with a constant amount of CBF1 expression vector pJH282 and increasing concentrations of either full-length NICD or the truncation proteins CDN1 and CDCN1T. Full-length NICD elevated I
B
protein expression in a dose-dependent manner, whereas by contrast neither CDN1 nor CDCN1T had any effect (Fig. 7E). The de-repression of I
B
gene transcription by NICD therefore results in enhanced expression of the NF-
B inhibitor.
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The Transcriptional Co-activator p300 Stimulates NICD-induced IB
Gene TranscriptionIt has been reported recently (53) that NICD activation of transcription operates via the recruitment of the transcriptional co-activator CBP/p300. Inhibition of transcription by p53 is an indicator of p300-dependent transactivation and has been reported previously (53) for Notch1-mediated transcription of artificial promoter constructs. HSC were co-transfected with the I
B
promoter-luciferase reporter, NICD, and increasing concentrations of a p53 expression vector. This experiment revealed that even the lowest concentration of p53 expression vector (100 ng) resulted in a complete repression of NICD-induced transcription (Fig. 8A). At higher concentrations of p53 we also observed a weaker inhibitory effect on basal I
B
promoter activity which probably reflects the ability of p53 to repress NF-
B-dependent transcription. Further support for a role for CBP/p300 was provided by a similar experiment in which HSC were co-transfected with the I
B
gene reporter, NICD, and increasing concentrations of an expression vector for p300. As shown in Fig. 8C, expression of p300 had the opposite effect to that observed with p53. p300 enhanced the level of NICD-induced I
B
promoter activity by at least 2-fold, and in many replicate experiments we observed up to a 4-fold enhancement. p300 was able to enhance I
B
promoter activity in the absence of NICD, which was expected from the fact that p300 is a co-activator of NF-
B-dependent transcription. However, the data in Fig. 8C indicate that NICD and p300 act synergistically to enhance I
B
gene transcription. Lack of significant attenuation of the activity of the internal control Renilla luciferase vector pRLTK (Fig. 8, B and D)in these experiments confirms the specificity of the effects of p53 and p300 on NICD-induced I
B
promoter activity.
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DISCUSSION |
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We have shown that CBF1 is the protein component of the previously uncharacterized high mobility NF-B protein-DNA complex in activated HSC (31). By introducing nucleotide base substitutions into the consensus NF-
B DNA-binding site used in our previous study, we were able to distinguish between protein-DNA interactions assembled by p50:p65 and CBF1. These studies revealed that CBF1 binds to a site that overlaps with the NF-
B-binding site. To date dual NF-
B/CBF1 sites that function as targets of CBF1-mediated transcriptional repression have only been described for a limited number of genes including IL-6, NF-
B2, and CYP2B1 (3740). We have shown that one (
B2 site) of five NF-
B/NF-
B-like sites in the human I
B
gene promoter is a functional dual NF-
B/CBF1-binding site. Overexpression of CBF1 resulted in repression of I
B
promoter activity in HSC; however, this effect was not observed when the CBF1-binding region of the
B2 site was mutated. Requirement for CBF1 DNA binding activity was further confirmed by showing that a CBF1 protein lacking DNA binding activity was unable to repress I
B
promoter activity. Taken together these data suggest that the
B2 site contains a functional CBF1-binding motif that can recruit CBF1 to the I
B
promoter where it acts as a transcriptional repressor. In order to assess the biological significance of CBF1-mediated repression of I
B
promoter function and to extend our observations to at least one other cell type, we carried out transfection studies with wild type and mutant CBF1 in COS1 cells. As COS1 cells are more efficiently transfected than primary rat HSC, we were able to assess the effects of CBF1 overexpression on the expression of endogenous I
B
protein. These experiments confirmed a powerful repressive effect of CBF1 on I
B
protein expression and also confirmed that a mutant CBF1 lacking DNA binding activity is unable to influence I
B
expression. In addition, EMSA analysis of NF-
B/CBF1 DNA binding activity revealed that overexpression of CBF1 in COS1 cells not only led to the expected increase in CBF1 DNA binding activity but also generated increased NF-
B (p50:p65) DNA binding activity. This latter observation led us to propose a model for the control of basal NF-
B activity by CBF1 (Fig. 9). At low levels of CBF1 expression, activity of the I
B
promoter will be relatively high leading to the maintenance of a large pool of inducible I
B
-bound NF-
B and low levels of active nuclear NF-
B. At higher levels of CBF1 expression, I
B
expression will be repressed which will lead to increased levels of constitutive NF-
B activity.
Several different mechanisms have been proposed for the inhibitory effects of CBF1 on gene expression. These include the potential for CBF1 to interact with components of the basic transcription machinery, inhibition of the binding of transcriptional activators at sites adjacent to the CBF1-binding motif, and recruitment of HDAC containing transcriptional co-repressor complexes (37, 39, 44, 5456). Several studies have shown that CBF1 can recruit at least two co-repressor-HDAC complexes as follows: the SMRT·NcoR·HDAC1 and CIR·HDAC2·SAP30 complexes (44, 55, 56). The HDACs catalyze removal of acetyl groups from histones (principally H3 and H4) and in doing so promote the formation of condensed chromatin structures at gene promoters that are less accessible to transcription factors (57). There is also evidence that HDACs have the potential to have direct effects on the activity of certain transcription factors via their ability to remove acetyl groups from these proteins (58). Although we have not ruled out a role for other potential mechanisms, we currently favor a role for HDACs as the mediators of repression of IB
gene transcription in response to CBF1. In support, we have shown that induction of CBF1 DNA binding in activated HSC was accompanied by a reduced level of acetylated H4 at the I
B
promoter and that Notch can reverse the repressive effects of CBF1. Notch signaling begins with DSL ligand binding, which then triggers a series of extracellular and intracellular proteolytic events that result in the release of NICD (59). Once in the nucleus, NICD is probably recruited to the CBF1 co-repressor complex via its interaction with the Ski-related protein (SKIP) (60) and the nuclear protein Mastermind (MAM) (61). SKIP associates with the CBF-1 co-repressor complex through its ability to bind to SMRT and CIR. Associations of SKIP with SMRT/CIR or NICD are mutually exclusive, and it has been demonstrated that even in the presence of a 4-fold excess of SMRT, NICD remains associated with a tri-protein complex inclusive of CBF1 and SKIP. NICD recruitment to SKIP and CBF1 therefore leads to destabilization of the SMRT-CBF1 interaction and loss of association of the SMRT-bound HDAC complex. Additional studies have shown that NICD can interact with histone acetyltransferases including PCAF, GCN5, and CBP/p300 (53, 62). One way in which NICD·CBF1 complexes can recruit histone acetyltransferase activity is via interaction with MAM, which in addition to stabilizing NICD/CBF1 binding to DNA induces the phosphorylation and recruitment of CBP/p300 to nuclear foci (61).
Our data show that activated HSC express Notch1 and Notch2, both of which are able to convert CBF1 to a transcriptional activator, by contrast expression of Notch3 which is an inhibitor of Notch1, and Notch2 signaling is lost during HSC activation (59). These findings coupled with our demonstration that co-culture of HSC with Jagged1-expressing L cells leads to induction of CBF1-dependent gene transcription indicates that activated HSC are responsive to Notch-DSL interactions. Furthermore, transfection of NICD increased IB
promoter activity in HSC and increased I
B
protein expression in CBF1 co-transfected COS1 cells. NICD activation of the I
B
promoter was dependent on CBF1. This was demonstrated by requirement for an intact CBF1 recognition sequence at the
B2 site, blockade of the effect by expression of a dominant negative CBF1 (RBP-J
R218H), and lack of transcriptional activation by truncated NICDs lacking the RAM domain required for interaction with CBF1. These findings were important in two respects. First, there are reports of CBF1-independent mechanisms of transcriptional activation by NICD, which we are able to rule out for I
B
(52, 63). Second, because it has been proposed that NICD converts CBF1 to a transcriptional activator by destabilizing association of CBF1 with SMRT/HDAC (60), our confirmation that NICD activates I
B
gene transcription via CBF1 supports our preference that CBF1 mediates repression of the I
B
promoter via recruitment of HDACs. Further support for a role for histone acetylation was provided by showing that overexpression of p53 inhibited NICD-induced I
B
promoter activity, whereas by contrast overexpression of p300 enhanced NICD-induced transcription.
Our observation that NICD can de-repress the IB
promoter and lead to an elevation of I
B
protein expression indicates that the Notch/CBF1 signaling pathway has the potential to attenuate NF-
B activity. We propose a model (Fig. 9) for the regulation of basal NF-
B activity by events described previously (4447, 55, 59, 60, 64, 65) for the CBF1-dependent Notch signal transduction pathway in both Drosophila and mammalian cells. In this model the interaction of Notch-expressing cells with cells expressing DSL family proteins results in the release of NICD and the replacement of HDAC with histone acetyltransferase (CBP/p300) activity at the I
B
promoter. This would lead to an elevation of I
B
protein expression and a reduction in the pool of active nuclear NF-
B. This provides a novel and potentially physiologically important pathway for altering NF-
B-dependent gene expression in a wide variety of mammalian cell types. With respect to the injured liver, DSL proteins are expressed by at least two types of liver cell, hepatocytes and macrophages, both of which have been shown to be in close juxtaposition to HSC (6668). It is therefore tempting to speculate that interaction of activated HSC with hepatocytes and macrophages may trigger Notch signaling and result in a diminution of NF-
B activity. Interaction of HSC with DSL expressing cells may therefore be an important determinant of the persistence and profibrogenic function of activated HSC.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| To whom correspondence should be addressed: Liver Group, Division of Infection, Inflammation and Repair, University of Southampton, Level D, Southampton General Hospital, Southampton SO16 6YD UK. Tel.: 44 2380796871; Fax: 44 2380794154; E-mail: dam2{at}soton.ac.uk.
1 The abbreviations used are: HSC, hepatic stellate cells; HDAC, histone deacetylase; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; IL, interleukin; FACS, fluorescence-activated cell sorter; NICD, Notch1 intracellular domain; ChIP, chromatin immunoprecipitation; wt, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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