The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 1934-1941
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kong, T.
Right arrow Articles by Colgan, S. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kong, T.
Right arrow Articles by Colgan, S. P.

Identification of Pur{alpha} as a New Hypoxia Response Factor Responsible for Coordinated Induction of the beta2 Integrin Family1

Tianqing Kong*, Melanie Scully{dagger}, C. Simon Shelley2,{ddagger} and Sean P. Colgan2,{dagger}

* Renal Division, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115; {dagger} Mucosal Inflammation Program, Division of Gastroenterology, University of Colorado Health Sciences Center, Denver, CO 80218; and {ddagger} Renal Division, Department of Medicine, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Central to the process of inflammation are hypoxic conditions that lead to the binding of circulating leukocytes to the endothelium. We have previously shown that such binding is mediated by monocytes being able to directly sense hypoxic conditions and respond by inducing their surface expression of the beta2 integrin family of adhesion molecules. In this study, we show that coordinated induction of the beta2 integrins during direct hypoxia-sensing occurs through transcriptional activation of each of the genes by which they are encoded. Certain of the molecular mechanisms that mediate this activation in transcription are dependent upon hypoxia-inducible factor-1 (HIF-1), whereas others are HIF-1 independent. In search of these HIF-1-independent mechanisms, we identified Pur{alpha} as a new hypoxia-response factor. Binding of Pur{alpha} to the HIF-1-independent beta2 integrin promoters is induced by hypoxia and mutagenesis of these Pur{alpha}-binding sites almost completely abolishes the ability of the promoters to respond to hypoxic conditions. Additional studies using siRNA directed against Pur{alpha} also revealed a loss in the hypoxic response of the beta2 integrin promoters. Taken together, our findings demonstrate that hypoxia induces a coordinated up-regulation in beta2 integrin expression that is dependent upon transcriptional mechanisms mediated by HIF-1 and Pur{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Acute and chronic inflammatory responses are characterized by significant shifts in tissue metabolism. As part of these changes, oxygen demand often exceeds supply and can result in overall oxygen deficits (hypoxia) (1, 2, 3, 4, 5). Central to the inflammatory process is the recruitment of large numbers of myeloid cells, such as neutrophils and monocytes (6). The vast majority of inflammatory cells are recruited to, as opposed to being resident at, inflammatory lesions. Use of existing oxygen by infiltrating leukocytes perpetuates the state of oxygen deficit experienced by the surrounding tissue (7, 8). Consequently, a thorough understanding of the molecular events that lead to myeloid recruitment at the sites of inflammation is an important step toward developing effective therapies.

It is now appreciated that adhesion-based interactions coordinated by leukocyte beta2 integrins are the primary means by which myeloid cells interact with other cell types, including endothelial and epithelial cells (9, 10). The beta2 integrins are a family of four glycoprotein heterodimers composed of a unique {alpha}-subunit, encoded by the CD11a, CD11b, CD11c, or CD11d gene, noncovalently associated with a common beta-subunit encoded by the CD18 gene (11, 12, 13, 14). Expression of the different subunits of the beta2 integrins is coordinately regulated during leukocyte differentiation and during activation. Such coordinated regulation occurs at the level of gene transcription (15).

We have previously shown that the hypoxia associated with inflammation dramatically induces transcription of the CD18 gene (16). The molecular mechanisms that mediate this induction involve the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1)3 (17, 18). Although this finding provided an initial insight into how hypoxia regulates beta2 integrin expression, the means by which hypoxia influences coordinated regulation of the different members of the beta2 integrin family remained unknown. In the current study, we aimed to identify these molecular mechanisms.

Initial observations revealed that hypoxia induces transcription, not only of the CD18 gene, but also each CD11 gene that encodes an {alpha}-subunit of the beta2 integrin family. Transcriptional induction of the CD11a and CD11d genes was found to be mediated by HIF-1-dependent mechanisms. However, induction of the CD11b and CD11c genes is mediated by mechanisms that are independent of HIF-1. Further studies revealed that hypoxia induces transcription of the CD11b and CD11c genes by mechanisms involving the ssDNA-binding protein Pur{alpha}. This finding identifies Pur{alpha} for the first time as a hypoxia-response factor. Furthermore, our results demonstrate that Pur{alpha} acts together with HIF-1 to drive coordinated induction of the beta2 integrin family in response to hypoxia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

The human promonocytic cell line U937 and human microvascular endothelial cells were obtained and cultured as previously described (16, 19). U937 cells were exposed to hypoxia by replacing the growth medium with fresh medium equilibrated with a hypoxic gas mixture and incubated in a hypoxic chamber (Coy Laboratory Products). Measured pO2 was 20 mm Hg with the balance made up of nitrogen, carbon dioxide (ambient 5% CO2), and water vapor (16). Where indicated, human microvascular endothelial cells monolayers were activated by the addition of 100 ng/ml LPS (List Biological Laboratories). Leukocyte-endothelial adhesion assays were performed as previously described (16, 20).

Immunoprecipitation and Western blotting

U937 cells were exposed to experimental conditions and washed with HBSS, and then their surface proteins were labeled with biotin as described previously (16, 21). Cells were lysed and debris was removed by centrifugation, and then lysates were precleared with protein-G Sepharose (Pharmacia Biotech) (16). CD18 immunoprecipitation was performed with 10 µg/ml of the mAb IB4 followed by incubation with protein-G Sepharose (16). Washed immunoprecipitates were boiled in nonreducing sample buffer, separated by SDS-PAGE under nonreducing conditions, and transferred to nitrocellulose (16). Biotinylated proteins were labeled with streptavidin-peroxidase and visualized by ECL (Amersham Biosciences). Densitometric analysis of resulting blots (n = 4 per experiment) was performed using Image J software (National Institutes of Health, Bethesda, MD).

In subsets of experiments, individual CD11 integrin chains were immunoblotted following U937 cell exposure to indicated periods of hypoxia. Anti-CD11a (clone MHM.24) was obtained from the Developmental Studies Hybridoma Bank, anti-CD11b polyclonal Ab was a gift from C. Parkos (Emory University, Atlanta, GA), anti-CD11c polyclonal Ab was obtained from Santa Cruz Biotechnology, and anti-CD11d was a gift from D. Staunton (ICOS, Bothell, WA).

Quantitative polymerase chain reactions

Messenger RNA was quantified by real-time PCR as described previously (22). The primer sets consisted of 1 µM of sense primer and 1 µM of antisense primer each containing SYBR Green I (Molecular Probes). Primer sets (sense sequence, antisense sequence, and product size, respectively) for the following genes were used: CD11a (5'-AACTGGACTCAGGATGCCC-3', 5'-CAAGGAAGGAACCAAGAGAGG-3', 239bp), CD11b (5'-TCTCAGAGTCCTTCTGTTAACAG-3', 5'-AGCTGAGGGGGGCTGGTGG-3', 299bp), CD11c (5'-GAGAAATGATCCCTCTTTGCC-3', 5'-GTCCTTTTGGGGAACACAGC-3', 230bp), CD11d (5'-TATCATGGATTCAACCTGG-3', 5'-CGGGCCACAGGCCAGGAG-3', 283bp), and human beta-actin (5'-GGTGGCTTTTAGGATGGCAAG-3', 5'-ACTGGAACGGTGAAGGTGACAG-3', 162bp). The analysis of beta-actin was used to control for variations in the quantity of starting template. Transcript levels and fold changes in mRNA were determined as described previously (23).

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed using U937 cells subjected to normoxia or hypoxia and the mAb 9C12 that specifically interacts with Pur{alpha} or control IgG (16, 24). Primer sets for the following genes were used in the PCR phase of the assays (sense sequence, antisense sequence, and product size, respectively): CD11a (5'-AGTGAGAAACCATGACAGCAGTG-3', 5'-GCGTCAGGAGGCCCGTGGG-3', 289bp), CD11b (5'-GGCTAAGTCTATTCAGCTTGTTCA-3', 5'-GGAACCACAAGGAAGCCACCAA-3', 275bp), CD11c (5'-TGCATCCATCTAAGCAAAGGGCA-3', 5'-GAGGAGTGCTGCCCTGGTCC-3', 278bp), CD11d (5'-CGGGGCTCCCTGGGTACCAA-3', 5'-CAGAAGGTGCTCACCCTCCCA-3', 250bp) and CD18 negative control (5'-TGCAACCCACCACTTCCTCCA-3', 5'-ACCCTCGGTGTGCTGGAGTC-3', 166bp).

Transfection assays

U937 cells were used to assess the induction of the beta2 integrin gene promoters by hypoxia. These cells were transfected by electroporation with a mixture of a plasmid that constitutively expresses Renilla luciferase (Invitrogen Life Technologies) and a construct generated by cloning one of the beta2 integrin gene promoters into the plasmid vector pATLuc such that it is immediately upstream of a firefly luciferase reporter. The CD11a promoter spanned nucleotides –525 to +103 relative to the major site of transcription initiation, the CD11b promoter spanned nucleotides –242 to +71, the CD11c promoter spanned nucleotides –128 to +36, the CD11d promoter spanned nucleotides –419 to +60, and the CD18 promoter spanned nucleotides –79 to +19 (15, 19, 25, 26, 27). Background firefly luciferase activity was assessed by transfecting U937 cells with the Renilla expression plasmid mixed with the parental plasmid pATLuc that lacks a functional beta2 integrin promoter (27). After transfection, cells were subjected to hypoxia or normoxia and lysed, and luciferase activity was assessed using a dual luciferase assay kit (Stratagene) and a Turner Designs luminometer. All firefly luciferase activity was normalized with respect to the constitutively expressed Renilla luciferase reporter gene. In subsets of experiments, Pur{alpha} binding site mutations were introduced into the CD11b and CD11c promoters by site-directed mutagenesis (28). Specifically, within the CD11b gene, the sequence 5'-GGCAGGCTG-3' spanning nucleotides –114 to –106 relative to major site of transcription initiation was mutated to the sequence 5'-GGCAttCTG-3' (28). The mutated nucleotides are indicated in lower case type. Within the CD11c gene the sequence 5'-CTTCCTTCCCC-3' spanning nucleotides –40 to –30 relative to major site of transcription initiation was mutated to the sequence 5'-aTTaaTTaaaa-3' (19). All mutations were confirmed by DNA sequencing. The ability of the mutated CD11b and CD11c promoters to respond to hypoxia was assessed as described above.

Inhibition of HIF-1{alpha}beta expression

HIF-1{alpha} depletion was accomplished by using phosphorothioate derivatives of antisense (5'-GCCGGCGCCCTCCAT-3') or control sense (5'-ATGGAGGGCGCCGGC-3') oligonucleotides as described previously (16). Western blot analysis for HIF-1{alpha} was performed as described previously (29). HIF-1beta repression was accomplished using commercial siRNA provided by Dharmacon.

Inhibition of Pur{alpha} expression

Pur{alpha} depletion was accomplished by using siRNA directed toward the Pur{alpha} mRNA sequence 5'-CCGCAAGTACTACATGGATCT-3'. As a control for these experiments, a mismatched siRNA was used with the sequence 5'-CCGCAAGTAtTACgTGGATCT-3'. The two mismatches to the Pur{alpha} mRNA sequence in this control siRNA are indicated in lowercase type. Both the test and control siRNA were expressed from the plasmid vector pSUPER provided by Dr. R. Agami of the Netherlands Cancer Institute (Amsterdam, The Netherlands) (30).

Expression of recombinant Pur{alpha}

The Pur{alpha} expression construct, pHAPur1, was provided by E. Johnson (Mount Sinai School of Medicine, New York) and the empty vector control, pHA, produced by religation following liberation of the Pur{alpha} sequence by RsrII and EcoRI digestion (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Leukocyte hypoxia induces increased adhesion to endothelia

Leukocyte beta2 integrins are coordinately expressed as heterodimers composed of a common CD18 subunit linked to a subunit of CD11a, CD11b, CD11c, or CD11d (11, 12, 13, 14). In previous studies, we found that expression of the beta2 integrin CD18 subunit is induced by hypoxia (16). Furthermore, we demonstrated that this induced expression contributes in a significant way to increased leukocyte binding to endothelium (16). However, CD18 induction would have no functional meaning without a concomitant induction of at least one of the beta2 integrin CD11 subunits. Consequently, we hypothesized that CD11 subunits are hypoxia inducible. To address this hypothesis, U937 cells were subjected to normobaric hypoxia for 24 h and then assessed for their ability to adhere to LPS-activated endothelial cells in the presence of inhibitory Abs directed against CD11a or CD11c. As shown in Fig. 1, the component of increased leukocyte adhesion attributable to hypoxia (3.1 ± 0.6-fold increase, p < 0.01) was nearly completely inhibited by Abs directed against either CD11a or CD11c, but was unaffected by an Ab directed against beta1 integrins. These results indicate that hypoxia-induced leukocyte adhesion to activated endothelial cells is dependent upon at least two of the beta2 integrin {alpha}-subunits.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 1. Leukocyte hypoxia enhances CD11-dependent adhesion to endothelia. The promyelocytic cell line U937 was exposed to normoxia (pO2 147 torr) or hypoxia (pO2 20 torr) for 24 h, labeled with the fluorescent marker BCECF, and then assessed for adhesion to LPS-activated endothelia. Adhesion assays were performed in the presence or absence of functionally inhibitory Abs directed against beta1 integrins (filled histograms), CD11a (open histograms), or CD11c (shaded histograms). Results are presented as the mean ± SEM of three independent experiments. Significant differences compared with when the anti-beta1 Ab is used are indicated at the levels of *, p < 0.05 and #, p < 0.01. Significant increases compared with normoxia are indicated at the level of **, p < 0.01.

 
Hypoxia induces surface expression of CD11 protein

Because hypoxia induces CD11-dependent adhesion, we reasoned that this might be mediated by increased expression of CD11 protein. Initially, we examined induction of the sum total of all five beta2 integrin molecules. This was achieved by subjecting U937 cells to a range of hypoxic periods (6–48 h), labeling surface proteins with biotin, and then immunoprecipitating CD18 from the lysates. Immunoprecipitates were resolved by SDS-PAGE and Western blots were probed with avidin-peroxidase. As can be seen in Fig. 2A, this protocol precipitates beta2 integrin heterodimeric complexes of CD11 and CD18. Consistent with our previous work, the common CD18 subunit is significantly induced by hypoxia (16), with induction observed as early as 12 h of hypoxia (2.7 ± 0.6-fold by densitometry, p < 0.05), and maximally at 48 h (9.7 ± 2.3-fold by densitometry, p < 0.01). In parallel, hypoxia-inducible CD11 protein is also observed, with maximal induction observed at 48 h of hypoxia (8.7 ± 1.9-fold by densitometry, p < 0.01). We next used immunoprecipitation and Western blot analysis to assess the expression of the individual members of the CD11 family (Fig. 2, B and C). These analyses revealed increased expression of each member of the CD11 family upon hypoxia exposure (for CD11a, CD11b, CD11c, and CD11d; 3.1 ± 0.4, 3.6 ± 1.1, 5.4 ± 1.3, and 5.5 ± 0.8-fold increase by densitometry, respectively, all p < 0.025). Consequently, these results support our hypothesis that all the proteins known to be formed into beta2 integrin heterodimers are coordinately induced by hypoxia.


Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 2. Hypoxia induces surface expression of beta2 integrin protein. A, U937 cells were exposed to hypoxia (pO2 20 torr) for the indicated periods of time. Cells were washed, and surface proteins were biotinylated and then lysed. CD18 was immunoprecipitated and resolved by SDS-PAGE, and the resultant Western blot was probed with avidin peroxidase. As indicated, the CD18 Ab immunoprecipitates heterodimeric complexes of CD11 and CD18. A representative experiment of four is shown. B, CD18 immunoprecipitations were immunoblotted with Abs directed against CD11a, CD11b, CD11c, or CD11d.

 
Hypoxia induces expression of CD11 mRNA

Our previous studies demonstrated that the increase in CD18 protein induced by hypoxia is reflected as an increase in the steady-state levels of CD18 mRNA (16). Consequently, we determined whether increased mRNA levels also underlie the induction of CD11 proteins elicited by hypoxia. Using real-time PCR, we observed that the steady-state level of each CD11 mRNA was increased in U937 cells exposed to hypoxia (Fig. 3A). For these purposes, CD18 was used as a positive control (16). Interestingly, both the time-course and magnitude of mRNA induction varied depending on the protein encoded. Maximal induction of both CD11b and CD11c mRNA occurred within 2 h of hypoxia exposure and then rapidly subsided. However, maximal induction of CD11a and CD11d occurred after 6 h, then subsided slowly. In addition, the maximal level to which hypoxia induced CD11d mRNA was approximately half of that to which it induced CD11a, CD11b, and CD11c mRNA.


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3. Hypoxia induces expression of beta2 integrin mRNA in U937 and PBMC. A, U937 cells were exposed to hypoxia (pO2 20 torr) for 0, 2, 6, or 12 h. The levels of mRNA encoding CD11a (filled histograms), CD11b (open histograms), CD11c (shaded histograms), and CD11d (stippled histograms) were then determined by real-time PCR relative to beta-actin. Results are presented as the mean ± SEM of three independent experiments. Significant increases compared with normoxia are indicated at the level of *, p < 0.01. B, PBMC were isolated and exposed to hypoxia (pO2 20 torr, 8 h). Total RNA was isolated and the levels of mRNA encoding CD11a, CD11b, CD11c, CD11d, and CD18 were then determined by real-time PCR relative to beta-actin. Results are presented as the mean fold change in hypoxia ± SEM of three independent experiments. Significant increases compared with normoxia are indicated at the level of *, p < 0.01.

 
To rule out that this response represents an abberancy in a leukemic cell line (U937 cells), anticoagulated blood from normal human volunteers was exposed to hypoxia (pO2 20 torr) for 8 h, PBMC were isolated, and assessed for CD11a-d expression by real-time PCR. As shown in Fig. 3B, these studies revealed a significant, hypoxia-inducible increase in all CD11 genes (4.1 ± 1.1, 3.4 ± 0.8, 5.2 ± 1.6, and 3.4 ± 0.5 for CD11a, b, c and d, respectively; all p < 0.025). Consistent with previous studies (16), CD18 served as a positive control for these experiments (6.1 ± 1.1-fold increase over normoxia, p < 0.025). Such results indicate that CD11 induction by hypoxia extends to cell types other than cultured leukocyte cell lines and into ex vivo tissue (whole human blood).

Transcriptional induction of the beta2 integrin gene promoters

We next determined whether hypoxia-inducible CD11 reflects an increase in transcription, as had been found previously with CD18 (16). As shown in Fig. 4A, U937 cells transiently transfected with the wild-type CD11a promoter (nucleotides –525 to +103) showed a 57 ± 7-fold increase in activity (p < 0.01, with mean normoxia luciferase values of 155 ± 35 counts) when subjected to 24 h of hypoxia. The CD11b promoter (nucleotides –242 to +71) showed a 43 ± 8-fold increase (p < 0.01, with mean normoxia luciferase values of 486 ± 68 counts), the CD11c promoter (nucleotides –128 to +36) showed a 31 ± 5-fold increase (p < 0.01, with mean normoxia luciferase values of 446 ± 49 counts), the CD11d promoter (nucleotides –419 to +60) exhibited a 16 ± 4-fold increase (p < 0.01, with mean normoxia luciferase values of 87 ± 7 counts) and, as previously shown, the CD18 promoter (nucleotides –79 to +19) exhibited a greater than 20-fold increase (16). These results demonstrate that transcription driven by the isolated promoter region of each beta2 integrin gene is induced by hypoxia. Thus, transcriptional activation of the beta2 integrin genes appears to help mediate increased leukocyte adhesion during conditions of hypoxia.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 4. Hypoxia induces beta2 integrin gene expression by both HIF-1-dependent and HIF-1-independent mechanisms (A). U937 cells were transfected with firefly luciferase reporter constructs containing the CD11a, CD11b, CD11c, CD11d, or CD18 gene promoters. Parallel transfections were performed using pATLuc that represents the parent of these constructs and is empty of beta2 integrin gene sequences. All transfection reactions contained the Renilla luciferase plasmid pRL-CMV. Transfected cells were exposed for 24 h to normoxia or hypoxia, and changes in the activity of pRL-CMV were taken as reflecting variations in transfection efficiency. Following correction for these variations, the level of firefly luciferase activity directed by the beta2 integrin constructs above that conferred by pATLuc under parallel conditions was determined. The fold increase in expression caused by hypoxia was then calculated. B, U937 cells were loaded with HIF-1{alpha} antisense or sense control oligonucleotides, as indicated, exposed to normoxia (Nx) or hypoxia (Hx), and examined for expression of HIF-1{alpha} by Western blot. C, U937 cells were loaded with HIF-1{alpha} antisense oligonucleotides (open histograms) or HIF-1{alpha} sense oligonucleotides (filled histograms). Loaded cells were then transfected with luciferase beta2 integrin promoter constructs in parallel with the parental vector pATLuc that is empty of beta2 integrin sequences. Cells were then exposed to hypoxia for 24 h and luciferase activity was determined. After correction for variations in pRL-CMV expression, the level of firefly luciferase activity directed by the beta2 integrin constructs above that conferred by pATLuc under parallel conditions was determined. The fold induction by hypoxia was then calculated. All histograms represent the mean ± SEM of three independent experiments, *, p < 0.01 and **, p < 0.05.

 
HIF-1-dependent and independent regulation of CD11 gene promoters

Each beta2 integrin promoter is activated by hypoxia (Fig. 2A). Our previous work demonstrated HIF-1{alpha}-dependent regulation of CD18 (16). Furthermore, chromatin immunoprecipitation analysis directly demonstrated HIF-1 binding to the endogenous CD18 gene (16). To determine whether CD11 promoters are controlled by HIF-dependent mechanisms, we first screened for expression of the various {alpha}-chains of HIF (HIF-1, 2, and 3) by Western blot. These studies revealed that U937 cells express only HIF-1, but not HIF-2 or HIF-3 (data not shown). Based on this evidence, we blocked expression of HIF-1{alpha} with antisense oligonucleotides (16) (Fig. 2B; 72 ± 6% decrease in protein, p < 0.025) and examined CD11a-d inducibility by hypoxia. HIF-1{alpha} antisense treatment resulted in 49 ± 6%, 44 ± 7%, and 88 ± 9% reduction in hypoxia-inducibility, respectively, for CD11a, CD11d, and CD18 promoters (Fig. 2C). Neither CD11b or CD11c promoter activity was significantly influenced by the HIF-1{alpha} antisense oligonucleotide, suggesting that both HIF-1-dependent and HIF-1-independent mechanisms regulate hypoxic induction of the beta2 integrins.

Additional evidence of a role for HIF-1 was provided by targeted repression of HIF-1beta. For these purposes, we used siRNA directed against HIF-1beta and screened CD11a-d and CD18 promoters. This siRNA approach effectively decreased the expression of HIF-1beta by 82 ± 7% by real-time PCR. Similar to our findings with HIF-1{alpha} antisense, HIF-1beta siRNA blocked induction of CD11a, CD11d, and CD18 (58 ± 8%, 63 ± 10%, and 78 ± 11% reduction in hypoxia-inducibility, respectively, all p < 0.025). CD11b and CD11c promoter activity were not significantly influenced by the HIF-1beta siRNA (p = not significant). Moreover, the combination of HIF-1{alpha} antisense and HIF-1beta siRNA to maximally block the HIF pathway did not influence this induction pattern (data not shown).

The transcription factor Pur{alpha} exhibits increased binding to the beta2 integrin gene promoters during hypoxia

Our use of HIF-1{alpha} antisense oligonucleotides and HIF-1beta siRNA indicate that hypoxia induces CD11b and CD11c gene expression by mechanisms that are, for the most part, independent of HIF-1 (Fig. 4B). Important in this regard, our own analysis of the individual CD11 gene promoters revealed no obvious consensus motifs for HIF (S. Colgan, unpublished observations). Consequently, in the next phase of our analysis, we sought to identify the HIF-1-independent mechanisms. We reasoned that coordinated induction of the beta2 integrin genes would likely involve common transcription factors. Therefore, we analyzed the beta2 integrin gene promoters for shared consensus transcription factor binding sites. This analysis revealed that all the CD11 gene promoters contain close repeats of the sequence GGN, in which N is not G. Such repeats represent the recognition element of the transcription factor Pur{alpha} (31, 32). Our own analysis has revealed that the CD18 gene promoter contains no binding site for Pur{alpha} (data not shown). ChIP analyses of each of the CD11 gene promoters demonstrated that binding of Pur{alpha} is indeed a common feature of the CD11 genes (Fig. 5). Furthermore, this analysis revealed that Pur{alpha} binding to the CD11 gene promoters is strongly induced by hypoxia (Fig. 5). For these purposes, the proximal region of the CD18 promoter served as a negative control.


Figure 5
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 5. Pur{alpha} binding to the CD11 gene promoters in vivo. Chromatin immunoprecipitation was performed to examine Pur{alpha} binding to the CD11 gene promoters in normoxic (N) and hypoxic (H) U937 cells. Lane 1, normoxia input genomic DNA; Lane 2, hypoxia input genomic DNA; Lane 3, normoxia Pur{alpha} immunoprecipitation; Lane 4, hypoxia Pur{alpha} immunoprecipitation; Lane 5, normoxia IgG control immunoprecipitation; Lane 6, hypoxia IgG control immunoprecipitation. The proximal CD18 gene promoter was used as a negative control. A representative example of three independent experiments is shown.

 
Role of Pur{alpha} in HIF-1-independent induction of the CD11b and CD11c genes

We next addressed the functional significance of Pur{alpha} to the HIF-1-independent induction of CD11. Given the possible interactions of HIF-1{alpha} and Pur{alpha} on the CD11a and CD11d promoters (Figs. 4 and 5, respectively), we focused on the CD11b and CD11c promoters. First, as shown in Fig. 6, mutagenesis of Pur{alpha} binding sites in the CD11b (Fig. 6A) and CD11c (Fig. 6B) promoters almost completely abolishes their induction by hypoxia. Importantly, the mutations did not adversely influence the basal activity of any of these promoters. These results suggest that Pur{alpha} binding sites contribute significantly to hypoxia induction that is HIF-1 independent. As a second approach, we knocked down expression of Pur{alpha} in U937 cells by siRNA. As shown in Fig. 7A, this strategy successfully decreased Pur{alpha} expression by >85% (based on densitometric determination). In addition, and consistent with our findings using ChIP (Fig. 5), this analysis revealed that hypoxia induces Pur{alpha} mRNA (Fig. 7A). Examination of CD11b and CD11c promoter activity in U937 cells treated with Pur{alpha} siRNA showed significantly decreased hypoxia-induction (Fig. 7, B and C), confirming the role of Pur{alpha} as a hypoxia response factor.


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 6. Binding of Pur{alpha} to the CD11b and CD11c gene promoters is necessary for their induction by hypoxia. The wild-type CD11b and CD11c promoters were introduced into the firefly luciferase reporter plasmid pATLuc to generate the constructs p11b-Wt and p11c-Wt, respectively. Point mutations that abolish Pur{alpha} binding were introduced into the wild-type sequences to generate the constructs p11b-{Delta}Pur and p11c-{Delta}Pur. U937 cells were transfected in parallel with p11b-Wt, p11b-{Delta}Pur, and pATLuc (A) or p11c-Wt, p11c-{Delta}Pur, and pATLuc (B). Each firefly luciferase plasmid was mixed with the Renilla luciferase plasmid pRL-CMV. Transfected cells were subjected to normoxia (filled histograms) or hypoxia (open histograms) for 24 h and assessed for firefly luciferase activity relative to Renilla luciferase. The relative level of firefly luciferase activity directed by p11b-Wt, p11c-Wt, p11b-{Delta}Pur, and p11c-{Delta}Pur above that conferred by the parent plasmid pATLuc was then calculated. Each histogram represents the mean ± SEM of three independent experiments.

 

Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 7. Pur{alpha} mediates hypoxia induction of the CD11b and CD11c promoters. A, U937 cells were transfected with a pSUPER plasmid that either expresses a siRNA directed against Pur{alpha} (Mch) or pSUPER expressing a mis-match control (M-Mch). Cells were then subjected to normoxia (N) or hypoxia (H) for 24 h and real-time PCR analysis used to determine the expression levels of Pur{alpha} and beta-actin mRNA. B, U937 cells were transfected in parallel with p11b-Wt and pATLuc each mixed with the Renilla luciferase plasmid pRL-CMV. Transfection of p11b-Wt was performed in the presence of a pSUPER plasmid that either expresses a siRNA directed against Pur{alpha} (Mch) or pSUPER expressing a mismatch control (M-Mch). Transfected cells were subjected to normoxia (filled histograms) or hypoxia (open histograms) for 24 h and luciferase activities determined. After correction for variations in pRL-CMV expression, the level of firefly luciferase activity directed by pATLuc and p11b-Wt was determined. Each histogram represents the mean ± SEM of three independent experiments. C, U937 transfections performed as described in B but using p11c-Wt rather than p11b-Wt.

 
Overexpression of Pur{alpha} recapitulates CD11b and CD11c promoter activity in hypoxia

Mutatagenesis and siRNA analysis suggested that Pur{alpha} binding is necessary for hypoxia induction of the CD11b and CD11c gene promoters (Figs. 6 and 7). We next sought to determine whether expression of Pur{alpha} was also sufficient to activate these promoters. This was achieved by cotransfecting the CD11b and CD11c reporter constructs with the plasmid pHAPur1 in which Pur{alpha} is constitutively expressed from the CMV promoter (24). As shown in Fig. 8A, transfection of U937 cells with pHAPur1 resulted in significant increases in Pur{alpha} expression in normoxia but not in hypoxia. Consistent with this engineered pattern of Pur{alpha} expression, CD11b and CD11c promoter activity was significantly increased under normoxic conditions but not under conditions of hypoxia. (Fig. 8, B and C). The levels to which pHAPur1 induced the CD11 promoters under normoxic conditions were approximately equivalent to those reached by the promoters under hypoxic conditions in the absence of Pur{alpha} overexpression. Taken together with our siRNA and mutagenesis results (Figs. 6 and 7), these observations indicate that Pur{alpha} is both necessary and sufficient to effect HIF-1-independent induction of the beta2 integrins in response to hypoxia.


Figure 8
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 8. Expression of recombinant Pur{alpha} is sufficient to recapitulate hypoxic induction of the CD11b and CD11c gene promoters. A, U937 cells were transfected with a plasmid that either constitutively expresses Pur{alpha} (pHAPur1) or is empty of Pur{alpha} cDNA (pHA). Cells were then subjected to normoxia (N) or hypoxia (H) for 24 h and real time PCR analysis used to determine the expression levels of Pur{alpha} and beta-actin mRNA. B, U937 cells were transfected in parallel with p11b-Wt and pATLuc each mixed with the Renilla luciferase plasmid pRL-CMV. Transfection of p11b-Wt was performed in the presence of either pHAPur1 that constitutively expresses Pur{alpha} or its empty equivalent pHA. Transfected cells were subjected to hypoxia for 24 h and firefly and Renilla luciferase assays performed. The levels of Renilla luciferase activity were taken as reflective of transfection efficiency and used to correct the firefly luciferase assay results. Each histogram represents the mean ± SEM of three independent experiments. C, U937 transfections performed as described in B but using p11c-Wt rather than p11b-Wt.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is recently appreciated that as part of the ongoing inflammatory response, hypoxia may be a contributing factor to the overall tissue phenotype (33, 34). Shifts in energy metabolism associated with either localized or systemic hypoxia represents both a cause and an effect of inflammation. Hypoxia promotes inflammatory lesions through mechanisms involving increased monocyte binding to the vascular endothelium (6), and the vast majority of inflammatory cells are recruited to, as opposed to being resident at, inflammatory lesions. This induced binding is dependent upon the increased function of adhesion molecules expressed on both the leukocyte surface and the surface of endothelium (35, 36, 37). A critical group of leukocyte adhesion molecules is the beta2 integrin family. This family comprises four heterodimers, composed of a common beta subunit encoded by the CD18 gene linked with one of four possible {alpha} subunits encoded by the CD11a, CD11b, CD11c, and CD11d genes (11, 12, 13, 14). In this study, we demonstrate that the {alpha} subunits of beta2 integrin family are coordinately induced by hypoxia and identify, for the first time, that Pur{alpha} represents a common transcriptional regulator of this response.

Previous work has shown that endothelial cells contribute to inflammation by directly sensing hypoxic conditions and responding through induction of the selectin and ICAM-1 genes (35, 36). These studies suggested that in hypoxia, endothelial cells communicate with leukocytes primarily through the release of chemokines/cytokines and the functional activation of leukocyte-adhesive properties (35, 36). However, it is recently appreciated that monocytes have the capacity to transcriptionally induce adhesion molecules (e.g., CD18) independent of the hypoxic response of the endothelium. Central to this mechanism is leukocyte HIF-1{alpha} (16). Because beta2 integrins are expressed on the monocyte surface as heterodimers, induction of the CD18 gene would have no functional consequence without concomitant induction of at least one of the CD11 genes. Initially, we profiled induction of the CD11a, CD11b, CD11c, and CD11d genes and demonstrated that each is induced at the mRNA and protein level following subjection to hypoxia. Notable were the differences in kinetics of this response for the various integrins. For example, induction of CD11b and CD11c is more rapid and transient than induction of CD11a and CD11d. Our previous studies have shown that the hypoxic induction of the CD18 gene is mediated by its interaction with HIF-1. Using antisense oligonucleotides and siRNA, we demonstrate in this study that HIF-1 contributes, albeit to a lesser extent than CD18, to induction of the CD11a and CD11d genes. However, this approach (HIF-1 antisense/siRNA) did not significantly influence hypoxia inducibility of CD11b or CD11c genes. Consequently, beta2 integrin induction by hypoxia appears to be mediated by both HIF-1-independent and HIF-1-dependent mechanisms. That said, we have no evidence that either CD11a or CD11d are directly regulated by HIF-1. For example, we did not identify an obvious consensus binding sequence for HIF-1{alpha}, and the degree of transcriptional inhibition using HIF-1 antisense approaches was significantly less that CD18, which bears a classic HIF response element (16). Such findings implicate a more indirect role for HIF in CD11a and CD11d regulation. From this standpoint, a number of transcription factors have been implicated in the control of beta2 integrin expression, including AP-1, MS-2, Sp1, and members of the Ets family (19, 27, 28, 38, 39, 40, 41, 42), and it is appreciated that HIF cooperates with a number of other transcriptional regulators (1, 43).

It is striking that the CD11a, CD11d, and CD18 genes that are dependent upon HIF-1 are all induced to maximal levels after the CD11b and CD11c genes that are HIF-1-independent. We reasoned that such HIF-1-independent mechanisms likely involved a transcription factor that binds both genes such that their induction can be coordinated. In this study, we identified this factor as Pur{alpha}. This is the first description of Pur{alpha} mediating a hypoxia response. However, previously a role for Pur{alpha} in inflammation had been implied by its control of TGF-beta1, CD11c, and CD43 expression and its increase in infiltrating eosinophils and activated endothelium during allergic reactions (19, 44, 45). Calcium mobilization is characteristic of the cellular activation that occurs during inflammation. In this regard, it is of note that the DNA binding activity of Pur{alpha} is increased by interaction with the calcium binding protein calmodulin (46).

Although Pur{alpha} has the capacity to bind dsDNA, its ability to bind ssDNA is at least 10-fold greater (31). The binding sites for Pur{alpha} within the CD11b and CD11c genes are associated with repeat sequences with the potential to form slippage structures with single-stranded loops. An induction in the availability of such loops might contribute to the dramatic induction of Pur{alpha} binding to the CD11b and CD11c promoters observed by chromatin immunoprecipitation. Indeed, it is possible that Pur{alpha} could itself contribute to the generation of single-stranded conformations because with isolated plasmids it can effect DNA unwinding (47).

Taken together, our results indicate that hypoxia coordinately induces beta2 integrins by mechanisms which involve both HIF-1{alpha} and Pur{alpha}. Our results indicate that indirect and direct oxygen sensing are linked at the molecular level. Specifically, while in this study we demonstrate that Pur{alpha} is required for direct hypoxia induction of the CD11c gene, previously we have reported it is also required for CD11c induction by phorbol ester (19). Taking phorbol ester as a mimic of a cytokine response, our data implicate Pur{alpha} in both direct and indirect hypoxia sensing during inflammation.


   Acknowledgment
 
We acknowledge Dionne Daniels for technical assistance on this work.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants HL60569, DK50189, and DE016191 from the National Institutes of Health, Grant-in-Aid 0355696T from the Northeast Affiliate of the American Heart Association, and by a grant from the Crohn’s and Colitis Foundation of America. Back

2 Address correspondence and reprint requests to Dr. Sean P. Colgan, Mucosal Inflammation Program, Division of Gastroenterology, University of Colorado Health Sciences Center, Denver, CO 80218 or Dr. Carl Simon Shelley, Massachusetts General Hospital, Department of Medicine, 149 13th Street, 8th Floor, Charlestown, MA 02129. E-mail addresses: sean.colgan{at}uchsc.edu or shelley{at}receptor.mgh.harvard.edu Back

3 Abbreviations used in this paper: HIF-1, hypoxia-inducible factor-1; ChIP, chromatin immunoprecipitation. Back

Received for publication January 11, 2007. Accepted for publication May 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Cummins, E. P., C. T. Taylor. 2005. Hypoxia-responsive transcription factors. Pflügers Arch. 450: 363-371. [Medline]
  2. Karhausen, J., V. H. Haase, S. P. Colgan. 2005. Inflammatory hypoxia: role of hypoxia-inducible factor. Cell Cycle 4: 256-258. [Medline]
  3. Murdoch, C., M. Muthana, C. E. Lewis. 2005. Hypoxia regulates macrophage functions in inflammation. J. Immunol. 175: 6257-6263. [Abstract/Free Full Text]
  4. Sitkovsky, M. V., D. Lukashev, S. Apasov, H. Kojima, M. Koshiba, C. Caldwell, A. Ohta, M. Thiel. 2004. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Annu. Rev. Immunol. 22: 657-682. [Medline]
  5. Walmsley, S. R., K. A. Cadwallader, E. R. Chilvers. 2005. The role of HIF-1{alpha} in myeloid cell inflammation. Trends Immunol. 26: 434-439. [Medline]
  6. Lewis, J. S., J. A. Lee, J. C. Underwood, A. L. Harris, C. E. Lewis. 1999. Macrophage responses to hypoxia: relevance to disease mechanisms. J. Leukocyte Biol. 66: 889-900. [Abstract]
  7. Haddad, J. J.. 2003. Science review: redox and oxygen-sensitive transcription factors in the regulation of oxidant-mediated lung injury: role for hypoxia-inducible factor-1{alpha}. Crit. Care 7: 47-54. [Medline]
  8. Saadi, S., L. E. Wrenshall, J. L. Platt. 2003. Regional manifestations and control of the immune system. FASEB J. 16: 849-856.
  9. Luscinskas, F. W., S. Ma, A. Nusrat, C. A. Parkos, S. K. Shaw. 2002. The role of endothelial cell lateral junctions during leukocyte trafficking. Immunol. Rev. 186: 57-67. [Medline]
  10. Zen, K., C. A. Parkos. 2003. Leukocyte-epithelial interactions. Curr. Opin. Cell Biol. 15: 557-564. [Medline]
  11. Arnaout, M. A.. 1990. Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75: 1037-1050. [Free Full Text]
  12. Mazzone, A., G. Ricevuti. 1995. Leukocyte CD11/CD18 integrins: biological and clinical relevance. Haematologica 80: 161-175. [Abstract/Free Full Text]
  13. Springer, T. A.. 1990. Adhesion receptors of the immune system. Nature 346: 425-430. [Medline]
  14. Van der Vieren, M., H. Le Trong, C. L. Wood, P. F. Moore, T. St John, D. E. Staunton, W. M. Gallatin. 1995. A novel leukointegrin, {alpha}dbeta2, binds preferentially to ICAM-3. Immunity 3: 683-690. [Medline]
  15. Shelley, C. S., E. P. Bottinger, M. A. Aranaout. 1992. Transcriptional Regulation of beta2 Integrins Elsevier Sciences Publishers, Amsterdam.
  16. Kong, T., H. K. Eltzschig, J. Karhausen, S. P. Colgan, C. S. Shelley. 2004. Leukocyte adhesion during hypoxia is mediated by HIF-1-dependent induction of beta2 integrin gene expression. Proc. Natl. Acad. Sci. USA 101: 10440-10445. [Abstract/Free Full Text]
  17. Schofield, C. J., P. J. Ratcliffe. 2004. Oxygen sensing by HIF hydroxylases. Nat. Rev. Mol. Cell Biol. 5: 343-354. [Medline]
  18. Semenza, G. L.. 2000. HIF-1 and human disease: one highly involved factor. Genes Dev. 14: 1983-1991. [Free Full Text]
  19. Shelley, C. S., J. M. Teodoridis, H. Park, O. C. Farokhzad, E. P. Bottinger, M. A. Arnaout. 2002. During differentiation of the monocytic cell line U937, Pur {alpha} mediates induction of the CD11c beta2 integrin gene promoter. J. Immunol. 168: 3887-3893. [Abstract/Free Full Text]
  20. Zünd, G., D. P. Nelson, E. J. Neufeld, A. L. Dzus, J. Bischoff, J. E. Mayer, S. P. Colgan. 1996. Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells. Proc. Natl. Acad. Sci. USA 93: 7075-7080. [Abstract/Free Full Text]
  21. LeBivic, A., X. R. Francisco, E. Rodriguez-Boulan. 1989. Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line. Proc. Natl. Acad. Sci. USA 86: 9313-9317. [Abstract/Free Full Text]
  22. Synnestvedt, K., G. T. Furuta, K. M. Comerford, N. Louis, J. Karhausen, H. K. Eltzschig, K. R. Hansen, L. F. Thompson, S. P. Colgan. 2002. Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 (HIF-1) mediates permeability changes in intestinal epithelia. J. Clin. Invest. 110: 993-1002. [Medline]
  23. Pfaffl, M. W.. 2001. A new mathematical model for relative quantification in real-time RT- PCR. Nucleic Acids Res. 29: E45[Medline]
  24. Johnson, E. M., P. L. Chen, C. P. Krachmarov, S. M. Barr, M. Kanovsky, Z. W. Ma, W. H. Lee. 1995. Association of human Pur{alpha} with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNA Pur{alpha} recognition element. J. Biol. Chem. 270: 24352-24360. [Abstract/Free Full Text]
  25. Bottinger, E. P., C. S. Shelley, O. C. Farokhzad, M. A. Arnaout. 1994. The human beta2 integrin CD18 promoter consists of two inverted Ets cis elements. Mol. Cell. Biol. 14: 2604-2615. [Abstract/Free Full Text]
  26. Shelley, C. S., M. A. Arnaout. 1991. The promoter of the CD11b gene directs myeloid-specific and developmentally regulated expression. Proc. Natl. Acad. Sci. USA 88: 10525-10529. [Abstract/Free Full Text]
  27. Shelley, C. S., O. C. Farokhzad, M. A. Arnaout. 1993. Identification of cell-specific and developmentally regulated nuclear factors that direct myeloid and lymphoid expression of the CD11a gene. Proc. Natl. Acad. Sci. USA 90: 5364-5368. [Abstract/Free Full Text]
  28. Farokhzad, O. C., C. S. Shelley, M. A. Arnaout. 1996. Induction of the CD11b gene during activation of the monocytic cell line U937 requires a novel nuclear factor MS-2. J. Immunol. 157: 5597-5605. [Abstract]
  29. Comerford, K. M., T. J. Wallace, J. Karhausen, N. A. Louis, M. C. Montalto, S. P. Colgan. 2002. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res. 62: 3387-3394. [Abstract/Free Full Text]
  30. Brummelkamp, T. R., R. Bernards, R. Agami. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553. [Abstract/Free Full Text]
  31. Bergemann, A. D., E. M. Johnson. 1992. The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication. Mol. Cell. Biol. 12: 1257-1265. [Abstract/Free Full Text]
  32. Bergemann, A. D., Z. W. Ma, E. M. Johnson. 1992. Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Mol. Cell. Biol. 12: 5673-5682. [Abstract/Free Full Text]
  33. Cramer, T., Y. Yamanishi, B. E. Clausen, I. Forster, R. Pawlinski, N. Mackman, V. H. Haase, R. Jaenisch, M. Corr, V. Nizet, et al 2003. HIF-1{alpha} is essential for myeloid cell-mediated inflammation. Cell 112: 645-657. [Medline]
  34. Karhausen, J. O., G. T. Furuta, J. E. Tomaszewski, R. S. Johnson, S. P. Colgan, V. H. Haase. 2004. Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis. J. Clin. Invest. 114: 1098-1106. [Medline]
  35. Dore-Duffy, P., R. Balabanov, T. Beaumont, M. A. Hritz, S. I. Harik, J. C. LaManna. 1999. Endothelial activation following prolonged hypobaric hypoxia. Microvasc. Res. 57: 75-85. [Medline]
  36. Faller, D. V.. 1999. Endothelial cell responses to hypoxic stress. Clin. Exp. Pharmacol. Physiol. 26: 74-84. [Medline]
  37. Yonekawa, K., J. M. Harlan. 2005. Targeting leukocyte integrins in human diseases. J. Leukocyte Biol. 77: 129-140. [Abstract/Free Full Text]
  38. Bush, T. S., M. St Coeur, K. K. Resendes, A. G. Rosmarin. 2003. GA-binding protein (GABP) and Sp1 are required, along with retinoid receptors, to mediate retinoic acid responsiveness of CD18 (beta2 leukocyte integrin): a novel mechanism of transcriptional regulation in myeloid cells. Blood 101: 311-317. [Abstract/Free Full Text]
  39. Chen, H. M., H. L. Pahl, R. J. Scheibe, D. E. Zhang, D. G. Tenen. 1993. The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-specific promoter activity. J. Biol. Chem. 268: 8230-8239. [Abstract/Free Full Text]
  40. Nicolaou, F., J. M. Teodoridis, H. Park, A. Georgakis, O. C. Farokhzad, E. P. Boettinger, N. Da Silva, P. Rousselot, C. Chomienne, K. Ferenczi, et al 2003. CD11c gene expression in hairy-cell leukemia is dependent upon activation of the proto-oncogenes Ras and JunD. Blood 2003: 6-12.
  41. Pahl, H. L., R. J. Scheibe, D. E. Zhang, H. M. Chen, D. L. Galson, R. A. Maki, D. G. Tenen. 1993. The proto-oncogene PU. 1 regulates expression of the myeloid-specific CD11b promoter. J. Biol. Chem. 268: 5014-5020. [Abstract/Free Full Text]
  42. Rosmarin, A. G., D. G. Caprio, D. G. Kirsch, H. Handa, C. P. Simkevich. 1995. GABP and PU. 1 compete for binding, yet cooperate to increase CD18 (beta2 leukocyte integrin) transcription. J. Biol. Chem. 270: 23627-23633. [Abstract/Free Full Text]
  43. Laderoute, K. R.. 2005. The interaction between HIF-1 and AP-1 transcription factors in response to low oxygen. Semin. Cell. Dev. Biol. 16: 502-513. [Medline]
  44. Shelley, C. S., N. Da Silva, J. M. Teodoridis. 2001. During U937 monocytic differentiation repression of the CD43 gene promoter is mediated by the single-stranded DNA binding protein Pur{alpha}. Br. J. Haematol. 115: 159-166. [Medline]
  45. Wei, E. Q., Y. Irie, C. H. Kuo, Y. Ding, S. Y. Niu, E. Do, N. Miki. 1998. A single stranded DNA-binding protein, ssCRE-BP/Pur{alpha}, in rat lung and its increase in allergic airway inflammation. Jpn. J. Pharmacol. 78: 419-427. [Medline]
  46. Kuo, C. H., E. Nishikawa, H. Ichikawa, T. Sadakata, S. Y. Niu, N. Miki. 1999. Calmodulin functions as an activator of Pur{alpha} binding to single-stranded purine-rich DNA elements (PUR elements). Biochem. Biophys. Res. Commun. 255: 406-411. [Medline]
  47. Wortman, M. J., E. M. Johnson, A. D. Bergemann. 2005. Mechanism of DNA binding and localized strand separation by Pur{alpha} and comparison with Pur family member, Purbeta. Biochim. Biophys. Acta 1743: 64-78. [Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kong, T.
Right arrow Articles by Colgan, S. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kong, T.
Right arrow Articles by Colgan, S. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS