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Originally published online as doi:10.1189/jlb.0704429 on October 20, 2004

Published online before print October 20, 2004
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(Journal of Leukocyte Biology. 2005;77:120-127.)
© 2005 by Society for Leukocyte Biology

JAK2 is necessary and sufficient for interferon-{gamma}-induced transcription of the gene encoding gp91PHOX

Renu Kakar*, Bryan Kautz* and Elizabeth A. Eklund{dagger},1

{dagger} The Feinberg School of Medicine and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, and Jesse Brown VA Medical Center, Chicago Lakeside Division, Illinois; and
* Birmingham Veteran’s Administration Hospital, Alabama

1 Correspondence: Northwestern University Medical School, 710 N. Fairbanks Ct., Olson 8524, Chicago, IL 60611. E-mail: e-eklund{at}northwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the inflammatory response, interferon-{gamma} (IFN-{gamma}) increases transcription of the gene encoding gp91PHOX, a respiratory burst oxidase component. This gene (referred to as the CYBB gene) is transcribed in phagocytic cells differentiated beyond the promyelocyte stage, and transcription continues until cell death. Previous investigations identified a positive regulatory element in the proximal CYBB promoter referred to as the hematopoiesis-associated factor 1 (HAF1)-cis element. This element is activated by a multiprotein complex, which includes the IFN consensus sequence-binding protein (ICSBP). Interaction of this complex with the HAF1-cis element requires ICSBP tyrosine phosphorylation, which is induced by IFN-{gamma} stimulation of phagocytic cells. Previous studies also identified a negative cis element in the CYBB promoter. This element is repressed by the homeodomain protein HoxA10. HoxA10 tyrosine phosphorylation, which occurs in response to IFN-{gamma}, decreases HoxA10 DNA binding and therefore repression of CYBB transcription. In these studies, we determine Janus tyrosine kinase 2 (JAK2) activation is necessary and sufficient for IFN-{gamma}-induced CYBB transcription in phagocytic cells and also for ICSBP and HoxA10 tyrosine phosphorylation. Consistent with these results, we find JAK2 activation is sufficient to induce ICSBP interaction with the HAF1 element and abolish HoxA10 binding to the CYBBrepressor element. Therefore, these findings provide direct demonstration of JAK2 dependence of IFN-{gamma}-induced CYBB transcription. In addition, these results identify a mechanism mediating this effect.

Key Words: respiratory burst • interferon regulatory factor • Hox protein • CYBB gene • phagocyte function • neutrophil • monocyte


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mature phagocytic cells are characterized by the ability to generate superoxide and other toxic-free radicals via the respiratory burst [1 ]. The catalytic unit of the respiratory burst oxidase is composed of several proteins including gp91PHOX and p22PHOX. Although expression of p22PHOX is ubiquitous, gp91PHOX expression is myeloid-specific and restricted to cells that have differentiated beyond the promyelocyte stage [2 ]. In mature phagocytes, inflammatory mediators, including interferon-{gamma} (IFN-{gamma}), further increase transcription of the gene encoding gp91PHOX (the CYBB gene) [2 ]. As gp91PHOX is the rate-limiting catalytic component [3 ], increased CYBB transcription increases respiratory burst capacity during the inflammatory response. In contrast, IFN-{alpha}/ß stimulation of mature phagocytic cells decreases CYBB transcription and down-regulates the inflammatory response [4 ].

IFN-{gamma} binding to the IFN-{gamma} receptor (IFN-{gamma}R) induces {alpha}-chain dimerization and subsequent ß-chain recruitment. As Janus tyrosine kinase 2 (JAK2) is associated with ß-chains, ligand binding brings multiple JAK2 proteins into proximity with each other. This results in JAK2 dimerization, autotyrosine phosphorylation, and activation [5 ]. Receptor multimerization also brings activated JAK2 into proximity with JAK1, resulting in JAK2-dependent JAK1 activation [5 ]. This activated receptor complex phosphorylates signal transducer and activator of transcription (Stat) factors and impacts gene transcription [5 ]. IFN-{gamma}-induced gene transcription influences a number of specific phagocyte functions (reviewed in ref. [6 ]). In addition to CYBB transcription, IFN-{gamma} induces transcription of genes encoding the high-affinity Fc receptor, nitric oxide synthetase, and vascular cell adhesion molecule 1 [6 ]. Tyrosine phosphorylation of IFN regulatory factors (IRFs) and activation of ras and mitogen-activated protein kinases are also induced by IFN-{gamma} [6 ].

Although JAK2 activation is the best-described event resulting from IFN-{gamma} stimulation, it is possible that previously undescribed pathways are also directly activated by this receptor-ligand interaction. Indeed, direct dependence on JAK2 has not been demonstrated for most IFN-{gamma}-stimulated events in phagocytic cells, including regulation of the CYBB gene or other genes discussed above. Ligand binding to the IFN-{alpha}/ßR activates JAK1 and Tyk2 kinases, also activating Stat proteins. As IFN{alpha}/ß decreases CYBB transcription, this suggests CYBBtranscription requires activation of JAK2, not JAK1. Alternatively, CYBB transcription may rely on unidentified proteins directly activated by the IFN-{gamma}R. Determination of JAK2 dependence of IFN-{gamma}-induced CYBB transcription is the focus of these investigations.

We previously identified a cis element in the proximal CYBB promoter, which is necessary for IFN-{gamma}-induced transcription [7 8 9 ]. This element, referred to as the hematopoiesis-associated factor 1 ("HAF1") element, interacts with a multiprotein complex that includes PU.1, IRF1, the IFN consensus sequence-binding protein (ICSBP), and the cyclic AMP response element-binding protein-binding protein (CBP) [7 8 9 ]. In previous investigations, we performed electrophoretic mobility shift assays (EMSA) with nuclear proteins from untreated myeloid cell lines and a HAF1-cis element probe. Under these conditions, we determined that PU.1 and IRF1 interact with this cis element forming the "HAF1 complex’ [8 , 9 ]. However, in EMSA with nuclear proteins from IFN-{gamma}-treated cells, the HAF1 complex is a heterodimer of PU.1 + IRF1 or a PU.1 + ICSBP [9 ]. Also, in EMSA with nuclear proteins from IFN-{gamma}-treated cells, this probe generates an additional protein, which includes PU.1, IRF1, ICSBP, and CBP complex (the "HAF1a complex") [8 , 9 ]. IFN-{gamma} alters these protein-DNA interactions by inducing ICSBP tyrosine phosphorylation, which increases ICSBP interaction with PU.1, IRF1, and CBP [9 ].

We also identified a cis element in the CYBB promoter, which is repressed by a multiprotein complex including HoxA10, Pbx1a, and histone deacetylase 2 (HDAC2) [10 , 11 ]. This cis element (referred to as the Hox/Pbx-binding element) interacts with these proteins in EMSA with nuclear proteins from untreated myeloid cells but not nuclear proteins extracted after IFN-{gamma} treatment [10 ]. Similar to our results above, we also found IFN-{gamma} induces HoxA10 tyrosine phosphorylation. As HoxA10 tyrosine phosphorylation decreases binding affinity for this repressor element, IFN-{gamma} decreases HoxA10 repression of the CYBB gene [10 , 11 ]. Kinases involved in IFN-{gamma}-induced phosphorylation of ICSBP and HoxA10 have not been identified.

Therefore, IFN-{gamma} induces ICSBP and HoxA10 tyrosine phosphorylation, which increases CYBB transcription. The current investigations are designed to determine whether these events are mediated by JAK2 versus unidentified intermediates activated by the IFN-{gamma}R. In these studies, we use the U937 myelomonocytic cell line as a model for IFN-{gamma}-induced CYBB transcription [7 ]. IFN-{gamma} treatment of U937 cells also increases ICSBP and HoxA10 tyrosine phosphorylation [9 , 10 ]. In these studies, JAK2 will be overexpressed in U937 cells, as previous investigations determined overexpressed JAK2 is autoactivated [12 ]. By increasing JAK2 concentration in the cell, overexpression is hypothesized to facilitate JAK2 dimerization and autophosphorylation [12 ]. A dominant-negative form of JAK2 (DN-JAK2) [13 ] will also be used in these investigations. This DN-JAK2 lacks kinase activity and is hypothesized to interact with endogenous JAK2 and block autophosphorylation. These studies will provide initial determination of the role of JAK2 in modulating CYBB transcription during the inflammatory response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and mutagenesis
Cloning of the proximal 450-bp proximal promoter fragment into the pCATE reporter vector has been described previously [2 ]. This reporter vector is commercially available (Stratagene, LaJolla, CA). The JAK2 mammalian expression vector (JAK2/pRc) was a kind gift of Dr. Stuart Frank (University of Alabama, Birmingham). A truncation mutant of JAK2 with 829 amino acids of the JAK2 sequence was generated by polymerase chain reaction (PCR). This has been described previously to function as a DN-JAK2 [13 ]. The PCR product was subcloned into the pRc vector and sequenced on both strands to verify no unintended mutations had been introduced.

Oligonucleotides
Oligonucleotides were synthesized by the Core Facility at the Lurleen B. Wallace Comprehensive Cancer Center at the University of Alabama. Oligonucleotides used in EMSA and representing CYBB promoter sequences have been described previously: cybbHAF1 (5'-ctgctgttttcatttcctcattggaagaagaagcatag-3') [7 ] and cybbHox/Pbx (5'-ttcagttgaccaatgattattagccaattttctgataaaa-3') [10 ]. The cybbHAF oligo binds the transcriptional activation complex, which includes PU.1, IRF1, ICSBP, and CBP [8 ]. The cybbHox/Pbx oligo binds the repression complex, which includes HoxA10 and Pbx1a [10 ].

Cell culture and IFN-{gamma} treatment
The human myelomonocytic cell line U937 [14 ] was obtained from Andrew Kraft (University of Colorado, Denver). Cells were maintained and differentiated as described [7 ]. For differentiation experiments, U937 cells were treated for 48 h with 400 U per ml human recombinant IFN-{gamma} (Roche, Indianapolis, IN) [7 8 9 10 11 ]. In some studies, IFN-{gamma}-treated U937 cells were also treated with 100 µM of the kinase inhibitor Tryphostin B42 (AG490) [15 ].

Transfection of myeloid cell lines
To generate stable transfectants, U937 cells growing at log phase (32x106) were electroporated with 50 µg plasmid DNA as described [7 ]. Plasmids used in these transfections were mammalian expression vectors (pRc) to express JAK2, DN-JAK2, or empty vector control. Stable transfectant pools were selected in G418, 1 mg/ml, as described previously [9 , 11 ]. Transfectant pools were selected instead of clones to compensate for possible integration site effects.

In other experiments, U937 cells were transiently transfected with reporter gene vectors, with the proximal 450 bp of the CYBB promoter (cybb-pCATE) or vector control (as described previously in ref. [8 ]), vectors to express JAK2, DN-JAK2, or empty vector control, and a plasmid to control for transfection efficiency (cytomegalovirus–ß-galactosidase, as described previously; ref. [7 ]). Transfectants were incubated 72 h, with and without IFN-{gamma} (400 U/ml) for the last 48 h. Reporter gene activity was determined as described previously [7 ].

Immunoprecipitation and Western blotting
U937 stably transfected cells were lysed under denaturing conditions, as described [9 ]. Cell lysates (500 µg) were immunoprecipitated with antibody to JAK2 (a kind gift of Dr. Stuart Frank, University of Alabama), ICSBP (Santa Cruz Biotechnology, Santa Cruz, CA), HoxA10 (BAbCo, Berkley, CA), or glutathione S-transferase antibody (Santa Cruz Biotechnology) as an irrelevant control. Immunoprecipitates were collected with Staph protein A Sepharose and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and Western blots sequentially probed with the 4G10 antiphosphotyrosine antibody (Upstate Biotechnology, Waltham, MA) followed by JAK2 antibody, ICSBP antibody, or HoxA10 antibody. None of these blotting antibodies detected proteins in Western blots of proteins from control, irrelevant antibody immunoprecipitation; therefore, these samples are not shown.

Isolation of RNA and Northern blotting
RNA was isolated by the guanidinium isothiocyanate procedure as described [16 ]. Total RNA was separated on denaturing formaldehyde agarose gels, transferred to nylon membrane, and probed with random primer-labeled probes, as described previously [8 ].

Isolation of nuclear proteins and EMSA
Nuclear extract proteins were prepared from U937 cells by the method of Dignam, with protease inhibitors (as described) [7 ]. In some experiments, U937 cells were differentiated with 400 U/ml IFN-{gamma} before nuclear protein isolation. Oligonucleotide probes were prepared, and EMSA and antibody supershift assays were performed, as described [7 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpressed JAK2 is activated in U937 myeloid cells, and DN-JAK2 blocks IFN-{gamma}-induced JAK2 activation
Previously, other investigators demonstrated JAK2 overexpression results in autophosphorylation (and therefore, activation) in a receptor-independent manner [12 ]. Also, JAK2 truncated at amino acid 829 has been shown to function as a DN-JAK2 [13 ]. Therefore, we generated stable transfectants of the U937 myeloid cell line with a vector to overexpress JAK2 (JAK2/pRc), DN-JAK2 (DN-JAK2/pRc), or empty vector control. For these experiments, stable pools were selected instead of clones to compensate for integration site effects. Two independent stable pools were analyzed for each of these constructs.

In initial investigations, we determined whether JAK2 activation is altered in these stable transfectants as determined by JAK2 tyrosine phosphorylation. Total cell lysate proteins were immunoprecipitated under denaturing conditions with a JAK2-specific antibody (or irrelevant antibody). Immunoprecipitates were analyzed by serially probing Western blots with antiphosphotyrosine and anti-JAK2 antibodies. Consistent with previous results [12 ], we found increased tyrosine phosphorylation of overexpressed JAK2 in comparison with endogenous JAK2 in U937 cells (Fig. 1 ). Abundance of tyrosine-phosphorylated JAK2 in untreated U937 cells overexpressing JAK2 was similar to abundance of endogenous, tyrosine-hosphorylated JAK2 in IFN-{gamma}-treated U937 cells. In contrast, we found that DN-JAK2 expression in IFN-{gamma}-treated U937 cells decreases tyrosine-phosphorylated JAK2 in comparison with IFN-{gamma}-treated control cells. In DN-JAK2-expressing, IFN-{gamma}-treated transfectants, the abundance of activated JAK2 was similar to the abundance of endogenous, activated JAK2 in untreated U937 cells. In these experiments, JAK2 did not immunoprecipitate with a control, irrelevant antibody from any of these lysates under any of these conditions (not shown).



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  		Figure 1. Overexpressed JAK2 is activated in U937 cells, and DN-JAK2 inhibits IFN-{gamma}-induced JAK2 activation. U937 cells were stably transfected with a vector to overexpress JAK2, DN-JAK2, or vector control. Lysate proteins from these cells were analyzed for JAK2 activation by anti-JAK2 immunoprecipitation (IP) and antiphosphotyrosine (anti-PY) Western blotting, as indicated. Overexpressed JAK2 is tyrosine-phosphorylated in U937 cells, and DN-JAK2 inhibits IFN-{gamma}-induced JAK2 tyrosine phosphorylation in these cells.

 
JAK2 activation is sufficient for gp91PHOX expression and is necessary for IFN-{gamma}-induced gp91PHOX expression in U937 myeloid cells
We first used these U937 stable transfectant cell lines to investigate the role of JAK2 activation in endogenous gp91PHOX expression. To determine if JAK2 activation is sufficient to induce gp91PHOX expression, total RNA was isolated from JAK2-overexpressing and control vector transfectants. To determine if JAK2 activation is necessary for IFN-{gamma}-induced gp91PHOX expression, RNA was also isolated from IFN-{gamma}-treated, DN-JAK2-expressing or control vector transfectants. Northern blots were analyzed for gp91PHOX and control {gamma}-actin mRNA expression. We found JAK2 overexpression in U937 cells increases gp91PHOX message abundance in comparison with control U937 cells (Fig. 2A ). This increase is similar to the induction of gp91PHOX expression by IFN-{gamma} treatment of control U937 cells. In contrast, overexpression of DN-JAK2 impairs induction of gp91PHOX expression in IFN-{gamma}-treated transfectants (Fig. 2A) .



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  		Figure 2. JAK2 activation is necessary and sufficient for gp91PHOX expression. (A) JAK2 activation increases gp91PHOX mRNA abundance in U937 cells, and DN-JAK2 overexpression inhibits IFN-{gamma}-induced gp91PHOX expression in these cells. Total cellular RNA was isolated from U937 transfectants overexpressing JAK2, DN-JAK2, or vector control. Expression of gp91PHOX was determined with or without IFN-{gamma} treatment as indicated. We found JAK2 overexpression increases gp91PHOX mRNA abundance in U937 cells, and DN-JAK2 overexpression blocks IFN-{gamma}-induced gp91PHOXmRNA expression. Expression of {gamma}-actin was also determined as a loading control. (B) The JAK2 inhibitor AG490 blocks IFN-{gamma}-induced gp91PHOX expression in U937 cells, which were differentiated with IFN-{gamma} with or without AG490 treatment as indicated. Northern blots of total cellular RNA were probed for gp91PHOX and {gamma}-actin control mRNA. Expression of gp91PHOX was decreased in IFN-{gamma}-treated cells in the presence of JAK2 inhibition. (C) JAK2 activation increases CYBBpromoter activity in U937 cells, and DN-JAK2 overexpression inhibits IFN-{gamma}-induced CYBBpromoter activity in these cells. U937 cells were cotransfected with a reporter construct containing 450 bp of the proximal CYBB promoter (CYBB/pCATE) or control vector (pCATE) and vectors to overexpress JAK2, DN-JAK2, or empty vector control. Transfectants were analyzed for reporter gene activity with or without IFN-{gamma} treatment. JAK2 overexpression significantly increased CYBB promoter activity. In contrast, overexpressed DN-JAK2 inhibited IFN-{gamma}-induced CYBBpromoter activity in these cells.

 
To confirm these results, we investigated the effect of the tyrosine kinase inhibitor AG490 on IFN-{gamma}-induced gp91PHOX expression in U937 cells. In hematopoietic cells, AG490 specifically inhibits JAK2 [16 ] and JAK3 [17 ]. In contrast to JAK2, JAK3 is not activated by the IFN-{gamma} or granulocyte macrophage-colony stimulating factor receptor but is activated by ligand binding to the interleukin-2, -4, and -7 receptors. Therefore, AG490 would relatively be expected to specifically inhibit JAK2 activity in IFN-{gamma}-treated myeloid cells. U937 cells were IFN-{gamma}-treated with or without AG490 at doses previously demonstrated to inhibit IFN-{gamma}-induced JAK2 activation [16 ]. Total RNA was isolated from these cells and analyzed by Northern blot for gp91PHOX and {gamma}-actin mRNA abundance (Fig. 2B) , as described above. Consistent with the results of DN-JAK2 expression, JAK2 inhibition with AG490 blocks IFN-{gamma} induction of gp91PHOX expression in U937 cells.

JAK2 overexpression induces CYBB promoter activation, and DN-JAK2 blocks IFN-{gamma}-induced CYBB promoter activity in U937 myeloid cells
We next used these transfectants to investigate the impact of JAK2 on the CYBBpromoter. In these experiments, we used a 450-bp CYBB promoter/reporter construct (cybb/pCATE), previously demonstrated to include cis elements necessary for IFN-{gamma}-induced CYBBtranscription in U937 cells [8 ]. This CYBB promoter sequence includes the positive HAF1-cis element and the Hox/Pbx-binding repressor element [8 , 10 ]. U937 stable transfectants were cotransfected with cybb/pCATE or empty vector, and the impact of JAK2 or DN-JAK2 overexpression on reporter gene activity was determined. We found JAK2 overexpression significantly increases CYBBpromoter activity in comparison with control expression vector transfectants (P<0.001, n=6; Fig. 2C ). In these experiments, JAK2 overexpression increases CYBB promoter activity almost as much as IFN-{gamma} treatment of control vector U937 transfectants (P=0.02, n=6). In contrast, DN-JAK2 expression antagonizes IFN-{gamma}-induced CYBB promoter activity in U937 cells. CYBBpromoter activity in DN-JAK2 transfectants was not significantly different with and without IFN-{gamma} treatment (P<0.001, n=6; Fig. 2C ). Neither overexpression of JAK2 or DN-JAK2 nor IFN-{gamma} treatment influenced reporter expression from empty vector control pCATE.

JAK2 activation is sufficient for ICSBP tyrosine phosphorylation and binding to the CYBB HAF1-cis element and is necessary for IFN-{gamma}-induced ICSBP tyrosine phosphorylation
Previously, we found ICSBP tyrosine phosphorylation is necessary for IFN-{gamma}-induced CYBB transcription via the HAF1-cis element [8 , 9 ]. Therefore, we used our model system to investigate the role of JAK2 on ICSBP tyrosine phosphorylation. Nuclear proteins from U937 stable transfectants were immunoprecipitated with an ICSBP antibody (under denaturing conditions), and immunoprecipitates were analyzed by Western blots probed with an antiphosphotyrosine antibody. We found JAK2 overexpression increases ICSBP tyrosine phosphorylation in comparison with control U937 transfectants (Fig. 3A ). Consistent with this, overexpression of DN-JAK2 decreases ICSBP tyrosine phosphorylation in IFN-{gamma}-treated U937 transfectants. ICSBP protein abundance was not altered in any of these conditions, consistent with our previous results [9 ]. ICSBP did not immunoprecipitate with control, irrelevant antibody from any of these cell lysates under any of these conditions (not shown).



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  		Figure 3. JAK2 is necessary and sufficient for ICSBP tyrosine phosphorylation in U937 myeloid cells. (A) JAK2 overexpression increases ICSBP tyrosine phosphorylation, and DN-JAK2 expression inhibits IFN-{gamma}-induced ICSBP tyrosine phosphorylation in U937 cells. Lysate proteins from U937 stable transfectants with JAK2, DN-JAK2, or empty vector were analyzed for ICSBP tyrosine phosphorylation by anti-ICSBP immunoprecipitation (IP) followed by antiphosphotyrosine (anti-PY) Western blotting. U937 transfectants were analyzed with or without IFN-{gamma} treatment as shown. (B) JAK2 overexpression in U937 cells induces formation of the HAF1a complex in EMSA with the HAF1-cis element. EMSA were performed with a synthetic oligonucleotide probe representing the CYBB HAF1-cis element and nuclear proteins from U937 stable transfectants with empty vector or JAK2 expression vector. Overexpression of JAK2 increases binding of the previously described HAF1a complex, which requires ICSBP tyrosine phosphorylation (as described in ref. [9 ]). (C) ICSBP antibody disrupts the HAF1 and HAF1a complexes in EMSA with the HAF1-cis element and nuclear proteins from JAK2-overexpressing U937 cells. EMSA were performed with a synthetic oligonucleotide probe representing the CYBB HAF1-cis element and nuclear proteins from U937 stable transfectants with JAK2. Binding reactions were preincubated with preimmune serum or anti-ICSBP antibody. The HAF1a complex (PU.1+IRF1+ICSBP+CBP) is completely disrupted by this antibody, and the HAF1 complex (PU.1+IRF1 or PU.1+ICSBP) is partly disrupted, consistent with previous results using nuclear proteins from IFN-{gamma}-treated U937 cells [8 ].

 
As JAK2 overexpression increases ICSBP tyrosine phosphorylation, we investigated whether it increases formation of the ICSBP-containing HAF1a complex. EMSA were performed with the HAF1-CYBB-cis element probe and nuclear proteins from U937 stable transfectants overexpressing JAK2 or vector control. In previous investigations, we characterized extensively proteins that interact with the HAF1-cis element in EMSA with nuclear proteins from U937 cells, with and without IFN-{gamma} treatment. In those experiments, proteins interacting with the HAF1-cis element were identified by EMSA with competitor oligonucleotides and with specific antibodies [7 8 9 ].

Previously, we found the HAF1-cis element binds two specific protein complexes in EMSA with nuclear proteins from U937 cells: PU.1 monomer, which migrates as a high-mobility complex, and a heterodimer of PU.1 + IRF1, which migrates as a lower mobility complex (the HAF1 complex) [7 8 9 ]. In EMSA with nuclear proteins from IFN-{gamma}-differentiated U937 cells, the HAF1-cis element probe binds PU.1, the HAF1 complex, and a lower mobility HAF1a complex [8 , 9 ]. The HAF1a complex includes PU.1, IRF1, ICSBP, and CBP [9 ]. In EMSA with nuclear proteins from IFN-{gamma}-treated U937 cells, the HAF1 complex is PU.1 + IRF1 or PU.1 + ICSBP (see ref. [8 ]). In the current investigations, we found JAK2 overexpression induces formation of the HAF1a complex (Fig. 3B) , similar to studies with nuclear proteins from IFN-{gamma}-treated U937 cells [9 ].

Additional EMSA were performed to determine whether the HAF1 and HAF1a complexes include ICSBP in JAK2-overexpressing cells. Nuclear proteins from U937 cells overexpressing JAK2 were preincubated with an ICSBP antibody (or preimmune serum) prior to incubation with the HAF1-cis element probe (Fig. 3C) . In these current studies, binding of the HAF1a complex was completely disrupted by ICSBP antibody, consistent with previous results using nuclear proteins from IFN-{gamma}-treated cells. Also consistent with these previous results, ICSBP antibody partly disrupted binding of the HAF1 complex in EMSA with nuclear proteins from JAK2-overexpressing cells. Preimmune serum did not disrupt any of these complexes.

JAK2 activation is sufficient for HoxA10 tyrosine phosphorylation and decreased binding to the CYBB Hox/Pbx-binding cis element and is necessary for IFN-{gamma}-induced HoxA10 tyrosine phosphorylation
Previously, we found that HoxA10 tyrosine phosphorylation decreases CYBB repression via the Hox/Pbx-binding cis element [10 ]. Therefore, we determined the impact of JAK2 on HoxA10 tyrosine phosphorylation. Similar to the experiments in the previous section, HoxA10 was immunoprecipitated from U937 stable transfectants and analyzed by probing Western blots with antibody to phosphotyrosine. We found JAK2 overexpression increases HoxA10 tyrosine phosphorylation in comparison with vector control in U937 transfectants (Fig. 4A ). In contrast, DN-JAK2 overexpression blocks IFN-{gamma}-induced HoxA10 tyrosine phosphorylation in U937 cells. HoxA10 did not immunoprecipitate with irrelevant control antibody from any of these lysates under any of these conditions (not shown).



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  		Figure 4. JAK2 is necessary and sufficient for HoxA10 tyrosine phosphorylation in U937 myeloid cells. (A) JAK2 overexpression increases HoxA10 tyrosine phosphorylation, and DN-JAK2 expression inhibits IFN-{gamma}-induced HoxA10 tyrosine phosphorylation in U937 cells. Lysate proteins from U937 stable transfectants with JAK2, DN-JAK2, or empty vector were analyzed for HoxA10 tyrosine phosphorylation by anti-HoxA10 immunoprecipitation (IP) followed by antiphosphotyrosine (anti-PY) Western blotting. U937 transfectants were analyzed with or without IFN-{gamma} treatment as shown. (B) JAK2 overexpression in U937 cells inhibits formation of the HoxA10-containing complex, which binds the CYBB repressor cis element in EMSA, which were performed with a synthetic oligonucleotide probe representing the CYBB Hox/Pbx-binding cis element and nuclear proteins from U937 stable transfectants with empty vector or JAK2 expression vector. Overexpression of JAK2 decreases binding of the previously described HoxA10/Pbx1a complex, which requires nontyrosine-phosphorylated HoxA10 [10 ]. (C) HoxA10 antibody disrupts the low-mobility complex binding the CYBB-negative cis element probe in EMSA with nuclear proteins from empty vector-transfected U937 cells. EMSA were performed with a synthetic oligonucleotide probe representing the CYBB Hox/Pbx-binding cis element and nuclear proteins from U937 stable transfectants with control vector. Binding reactions were preincubated with preimmune serum or HoxA10 antibody. Consistent with previous results [10 ], the complex binding this probe is cross-immunoreactive with HoxA10.

 
As HoxA10 tyrosine phosphorylation decreases binding to the CYBB Hox/Pbx-binding cis element, we investigated the effect of JAK2 overexpression on HoxA10 DNA binding. Nuclear proteins from U937 stable transfectants with JAK2 expression vector or vector control were used in EMSA with the Hox/Pbx CYBB-cis element probe. We found abundant HoxA10/Pbx1 complex binding this probe in EMSA with control vector nuclear proteins, consistent with previous results [10 ]. In contrast, we found JAK2 overexpression decreases binding of this HoxA10-containing complex to the CYBB-cis element probe (Fig. 4B) . This is similar to the effect of IFN-{gamma} treatment in our previous experiments [10 , 11 ].

Additional experiments were performed to verify that HoxA10 interacts with this probe in EMSA with nuclear proteins from vector control cells. Nuclear proteins from U937 cells transfected with control vector were preincubated with HoxA10 antibody (or preimmune serum) prior to incubaton with the Hox/Pbx-binding CYBB probe (Fig. 4C) . Consistent with our previous results, the low-mobility complex binding this probe is cross-immunoreactive with HoxA10. In contrast, this complex is not disrupted with preimmune serum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IFN-{gamma} increases transcription of the CYBB gene in mature myeloid cells during the inflammatory response. Our previous investigations indicate this requires IFN-{gamma}-induced tyrosine phosphorylation of the transcription factors ICSBP and HoxA10. In this report, we find receptor-independent activation of JAK2 is sufficient to induce tyrosine phosphorylation of ICSBP and HoxA10 and transcriptional activation of the CYBB promoter. Using a DN-JAK2 and a JAK2 chemical inhibitor, we also find JAK2 activation is necessary for IFN-{gamma}-induced CYBB transcription and tyrosine phosphorylation of ICSBP and HoxA10.

Previously, we determined that ICSBP tyrosine phosphorylation is required for CYBB transcriptional activation via the HAF1-cis element. PU.1 and IRF1 bind this cis element in unstimulated cells, but ICSBP interaction with DNA-bound PU.1 and IRF1 requires tyrosine phosphorylation of specific ICSBP residues [9 ]. CBP is functionally necessary for IFN-{gamma}-induced CYBB transcription, and recruitment of CBP to the HAF-cis element depends on interaction with tyrosine-phosphorylated ICSBP [9 ]. In these studies, we find activated JAK2 is sufficient to induce assembly of this DNA-bound, transcriptional-activation complex.

Our previous investigations also determined that HoxA10 tyrosine phosphorylation impacts CYBB promoter function. We found that HoxA10, Pbx1a, and HDAC2 interact with a proximal CYBB promoter element and repress transcription. This repression requires histone deacetylase activity [18 ] but is abolished by HoxA10 tyrosine phosphorylation in response to IFN-{gamma} [10 ]. Phosphorylation of two HoxA10-homeodomain tyrosine residues decreases HoxA10 binding to the CYBB-cis element, disassembling the repressor complex [11 ]. In the current studies, we find activated JAK2 is adequate to decrease binding affinity of this HoxA10/Pbx1a/HDAC2 complex to the CYBB promoter. This provides another mechanism by which JAK2 impacts oxidase gene regulation.

Interaction between IFN-{gamma} and the IFN-{gamma}R induces JAK2 activation and many downstream events. However, absolute dependence of many of these events on activated JAK2 has not been demonstrated. For example, it was not known whether interaction between the IFN-{gamma}R and its ligand induces CYBB transcription via JAK2 or via a previously undescribed JAK2-independent signaling pathway. Therefore, the current investigations represent the first demonstration that JAK2 activation is necessary and sufficient for IFN-{gamma}-induced CYBB transcription. These investigations also provide the first demonstration of the absolute dependence on JAK2 of HoxA10 and ICSBP tyrosine phosphorylation, very important events in modulating gene transcription during the inflammatory response.

However, these results do not imply ICSBP or HoxA10 are substrates for JAK2. It is possible that JAK2 activates an intermediate kinase, which directly phosphorylates these proteins. Additionally, JAK2 activation may alter function of tyrosine phosphatases, thereby influencing the ICSBP or HoxA10 phosphorylation state. For example, we previously demonstrated that ICSBP and HoxA10 are substrates for Src homology-2-containing tyrosine phosphate 1 (SHP1) protein tyrosine phosphatase (PTP) [9 , 11 ]. Functional modulation of SHP1-PTP activity could be an alternative mechanism for the impact of JAK2 on ICSBP and HoxA10 phosphorylation. These various possibilities are currently under investigation in the lab.

Increased gp91PHOX expression increases the capacity for respiratory burst activity, mediating an important component of the innate immune response. However, production of superoxide and other free radicals via the respiratory burst results in a number of pathological states, including adult respiratory distress syndrome and reperfusion injury after vascular infarction. In these studies, we determine JAK2 is a crucial regulator of oxidase gene expression and therefore, free radical damage in response to inflammatory mediators. As JAK2 inhibitors are under development for clinical use, our studies suggest such agents could rationally be used to prevent tissue damage in pathological conditions with a sustained inflammatory response.


   ACKNOWLEDGEMENTS
 
This work was supported by a Veterans Administration Merit Review and NIH Grants R01 CA95266 and R01 CA90870 (E. A. E.). We thank Drs. Andrew Kraft, Joseph Biggs, and Stuart Frank for helpful comments and discussions.

Received July 29, 2004; revised September 15, 2004; accepted September 30, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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