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Cell Growth & Differentiation Vol. 12, 211-221, April 2001
© 2001 American Association for Cancer Research

ß-Chemokine Induction of Activation Protein-1 and Cyclic AMP Responsive Element Activation in Human Myeloid Cells1

Yang-Ming Yang, William C. Hatch, Zhong-Ying Liu, Bin Du2 and Jerome E. Groopman2,, 3

Divisions of Experimental Medicine, [Y-M. Y., W. C. H., Z-Y. L., J. E. G.] and Infectious Diseases [B. D.], Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02115


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Chemokines effect leukocyte chemotaxis and activation through their binding to specific G protein-coupled receptors. Although early steps in chemokine signal transduction pathways have been characterized, there is relatively limited information available at the transcription factor level. To that end, we have examined the binding activity on activation protein-1 (AP-1) and cyclic AMP responsive element (CRE) target sequences in human THP-1 myeloid cells after treatment with the ß-chemokine macrophage inflammatory protein (MIP-1{alpha}). MIP-1{alpha} induced both AP-1 and CRE activation. Although inhibition of protein kinase C blocked the AP-1 activity induced by this chemokine, there was no decrease in CRE activation in the presence of a protein kinase A inhibitor. Using kinase assays, it appeared that mitogen-activated protein kinase pathways were involved in CRE activation. In addition, HIV-1 infection of THP-1 cells resulted in constitutive activation of AP-1 and CRE elements but no further response to MIP-1{alpha} treatment. These results suggest that ß-chemokines act via protein kinase C-dependent pathways and mitogen-activated protein kinase pathways to modulate the host transcriptional response in myeloid cells, and that this response is altered by HIV infection.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Chemokines are low molecular weight proteins initially recognized as inflammatory mediators with potent chemotactic effects on a variety of myeloid cell types (1, 2, 3, 4, 5) . This family of molecules is subdivided into two major groups based on the presence or absence of an amino acid between the first two cysteines: the {alpha}-chemokines structured as C-X-C and the ß-chemokines as C-C. Both {alpha}- and ß-chemokines appear to modulate cell proliferation and to direct cell migration. In addition to the release of chemokines as part of the host response in inflammation, the phenomenon of molecular piracy of chemokines by herpes viruses has been recognized recently. The ß-chemokine MIP4 -1 as well as the receptor for the {alpha}-chemokine IL-8, are homologous to open reading frames of the human herpes virus 8/Kaposi’s sarcoma herpes virus (6, 7, 8, 9, 10) . This observation suggests that virus-encoded chemokine homologues may be important in the pathology of herpetic infections as well as in the genesis of associated neoplasms such as Kaposi’s sarcoma.

With the emerging role of chemokines in both physiological and pathological processes, attention has focused on how these low molecular weight proteins may accomplish their pleiotropic biological effects. Chemokine receptors are members of the group of seven-transmembrane receptors. These receptors are functionally linked to phospholipases via heterotrimeric G proteins, with downstream generation of inositol triphosphate resulting in intracellular Ca2+ release, Ca2+ channel opening, and protein kinase C activation (1) . Signaling by the IL-8 (11) , formylmethionylleucylphenylalanine (12) , and MCP-1 (13) receptors is sensitive to inhibition by pertussis toxin, indicating that these receptors are coupled to a Gi protein. Gi inhibition of adenyl cyclase has been shown to follow chemokine receptor stimulation. Cotransfection experiments revealed that Gai was coupled to CCR1, a ß-chemokine receptor for MIP-1{alpha} and RANTES as well as for MCP-1. Signaling studies of the ß-chemokine receptors in transfected HEK-293 cells revealed potent, agonist-dependent inhibition of adenyl cyclase and mobilization of intracellular calcium (13) , consistent with receptor coupling to Gai. However, the downstream effects of chemokine-mediated inhibition of adenyl cyclase and calcium release in leukocytes are not well characterized. In particular, very little is known regarding modulation of nuclear transcription factors by {alpha}- and ß-chemokines.

Two of the major classes of regulatory elements that contribute to transcriptional regulation by extracellular signals are the AP-1/TRE (14 , 15) and ATF/CRE (16 , 17) sequence motifs. The AP-1/TRE element (TGACTCA) was originally defined as the AP-1 binding site or the TRE. The ATF/CRE element (TGACGTCA) was defined as the ATF binding site or CRE. Several proteins have now been shown to bind to these cis-acting elements. A group of proteins, including those encoded by the Jun and Fos families (18, 19, 20, 21, 22, 23, 24) , recognizes the AP-1/TRE site. Growth factors, cytokines, T-cell activators, neurotransmitters, and UV irradiation (25, 26, 27) can induce these proteins. The ATF/CRE site is recognized by a family of proteins referred to as ATF or CREB (28, 29, 30, 31, 32, 33, 34, 35, 36) . This family of proteins has been implicated in cAMP-, calcium- and virus-induced alterations in transcription (37, 38, 39) . These two families, based on their protein structures, belong to the basic region/leucine zipper superfamily of transcription factors. The proteins in this basic region/leucine zipper superfamily can form intra- or interfamily heterodimers. These two families of transcription factors are directly downstream of the cellular second messengers, cAMP and calcium, which have been shown to be mobilized by certain chemokines (2) . Nevertheless, to our knowledge, direct data on transcriptional regulation by ß-chemokines are not available.

We have investigated the regulation of nuclear transcription factors by the ß-chemokine MIP-1{alpha} in THP-1 myeloid cells and found that activation of both AP-1 and CRE was induced by this ß-chemokine. This activation was mediated by PKC and MAP kinases but not by PKA. Moreover, HIV infection of THP-1 cells significantly altered the transcriptional response to exogenous MIP-1{alpha}, indicating that part of the host inflammatory response mediated by chemokines may be blunted after infection with this retrovirus.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
MIP-1{alpha} Induces Dose-dependent Binding Activity to AP-1 and CRE Target Sequences.
To determine whether chemokines affect the transcription of genes regulated by AP-1 and CRE, nuclear proteins were prepared from THP-1 monocytic cells treated with the ß-chemokine, MIP-1{alpha}. The binding activity of these nuclear proteins to AP-1 and CRE target sequences was tested with EMSA. Fig. 1Citation (A-I and A-II) demonstrates that 30 nM MIP-1{alpha} can specifically stimulate AP-1 or CRE activation. The 10x unlabeled AP-1 or CRE sequence completely competed the binding activity, whereas the 10x unlabeled mutant AP-1 or CRE sequence as well as the 10x extra nonspecific DNA sequence did not change the binding activity of either AP-1 or CRE. When anti-MIP-1{alpha} antibody was added to the culture medium of THP-1 cells before MIP-1{alpha} stimulation, the activation of AP-1, and CRE induced by MIP-1{alpha} was blocked (Fig. 1Citation , Lane 7 in panels A-I and A-II). It is interesting that the UV-responsive element (40, 41, 42) , which is homologous to AP-1 (with a 3-bp difference) and CRE (with a 2-bp difference), shared binding proteins with the CRE sequence but not with the AP-1 sequence in THP-1 cells. This phenomenon contrasts with what we observed in keratinocytes (27) .



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Fig. 1. A, MIP-1{alpha} induces sequence-specific binding to AP-1 and CRE probes on THP-1 cells. Nuclear proteins (5 µg) prepared from THP-1 cells 30 min after mock (Lane 1) or chemokine (Lanes 2–7) treatments were incubated with 0.4 ng of 32P-labeled AP-1 (panel A-I) or CRE (panel A-II) sequence in the presence of DNA-binding buffer for 10 min at room temperature, followed by separation on 7.5% PAGE and analysis via autoradiography. The labels (above Lanes 3–6) indicate the nonradiolabeled oligonucleotides used for the preincubation. The amount of each oligonucleotide was 10-fold (4 ng) of the labeled target sequence and was added 10 min before addition of the 32P-labeled probe. Lane 7 shows the nuclear protein from cells that were pretreated with anti-MIP-1{alpha} antibody. Arrows, position of the protein-AP-1 or protein-CRE complexes. B, MIP-1{alpha} induces AP-1 and CRE activities in THP-1 cells in a dose-dependent manner. Nuclear proteins (5 µg) extracted from THP-1 cells 30 min after mock or chemokine treatments were incubated with the indicated 32P-labeled target sequence. Lane 1 is the nuclear protein prepared from control THP-1 cells, and Lanes 2–4 are the proteins from the cells treated with MIP-1{alpha}. C, kinetics of MIP-1{alpha}-induced AP-1 and CRE activation. Nuclear proteins were isolated from THP-1 cells 0–2 h after MIP-1{alpha} treatment.

 
To demonstrate the dose dependence and dynamics of MIP-1{alpha} regulating AP-1 and CRE binding activity, THP-1 cells were treated with different concentrations of MIP-1{alpha} for different time periods. The binding activity of the nuclear proteins from the THP-1 cells to AP-1 and CRE target sequences was tested with EMSA. The data in Fig. 1Citation (B-I) revealed that 10 nM MIP-1{alpha} can weakly stimulate AP-1 activation, with saturation of AP-1 binding activity at a concentration of 30 nM. AP-1 activation appeared as early as 30 min after MIP-1{alpha} treatment, and elevated AP-1 activity was maintained up to 2 h (Fig. 1Citation , C-I). Compared with AP-1, the CRE sequence showed a higher binding activity upon stimulation by 10 nM MIP-1{alpha}, with activation increasing up to 100 nM (Fig. 1Citation , B-II). The high binding activity to CRE was observed by 30 min and peaked at 1 h. After 1 h, the CRE activation decreased, although at 2 h the binding activity was still significantly higher than the control (Fig. 1Citation , C-II).

Transcriptional Activation Induced by MIP-1{alpha} via AP-1 and CRE Sequences.
As a measure of the transcriptional activation in THP-1 cells induced by MIP-1{alpha}, the CAT construct, driven by a 5x AP-1 or a 3x CRE sequence was transfected into THP-1 cells together with a ß-gal expression plasmid via electroporation. Thirty-six h after transfection, the cells were treated with MIP-1{alpha}, and total protein extracts were prepared 48 h after electroporation (12 h after MIP-1{alpha} was added). To correct for varying transfection efficiencies, MIP-1{alpha}-induced transcriptional activities were normalized based on ß-gal activities. As demonstrated in Fig. 2Citation , before MIP-1{alpha} treatment AP-1 or CRE-driven transcriptional activity in THP-1 cells was similar to the background (the transcriptional activity in cells transfected by the vector without a promoter). Both AP-1 and CRE-mediated transcriptional activation showed increases in binding activity after the MIP-1{alpha} treatments. The AP-1-CAT activity induced by MIP-1{alpha} was much higher than the CRE-CAT activity induced by this chemokine (Fig. 2)Citation . This difference is perhaps attributable to the differences in plasmid construction; AP-1-CAT was driven by a pentamer and CRE-CAT was driven by a trimer.



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Fig. 2. Transcriptional activity mediated by AP-1 and CRE. The CAT vector without promoter, AP-1-CAT or CRE-CAT, was transfected into THP-1 cells, and MIP-1{alpha} was added to the transfectants, as labeled on the X axis. Y axis, values obtained in the CAT assays, which were quantified with the aid of a phosphorimager as the acetyl form divided by the total radioactivity and normalized per basal level of the control activity and per transfection efficiency based on ß-gal activity. The CAT activity from AP-1-CAT transfectants or CRE-CAT transfectants without MIP-1{alpha} treatment was used as the control. Data are the results from three independent transfections; bars, SD.

 
Protein Kinase Pathways Involved in MIP-1{alpha}-induced Binding to AP-1 and CRE.
To determine the signaling mechanism through which AP-1 and CRE DNA binding activity may be induced by MIP-1{alpha}, Rp-cAMP and staurosporine were used to selectively block the adenyl cyclase pathway and PKC pathway, respectively. As shown in Fig. 3Citation (A-I), THP-1 cells treated with staurosporine, at a specific PKC inhibition concentration of 10-7 M or 10-9 M (Lanes 5 and 6, respectively; Refs. 43 and 44 ), exhibited a significantly decreased AP-1 response to MIP-1{alpha} stimulation, whereas Rp-cAMP treatment showed no effect on the AP-1 sequence (Lanes 3 and 4). On the other hand, as shown in Fig. 3Citation (A-II), Rp-cAMP-treated THP-1 cells (45 , 46) showed no change in binding activity to the CRE sequence after MIP-1{alpha} stimulation, even with increased doses (10 and 50 nM, Lanes 4 and 3, respectively). It is interesting that a high concentration (10-7 M) of staurosporine blocked the MIP-1{alpha}-induced binding activity to CRE (Lane 5), whereas a very low concentration (10-9 M) of staurosporine, which is more specific for PKC, resulted in a weaker inhibition of binding activity to CRE.



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Fig. 3. MIP-1{alpha} induces AP-1 activation through the PKC signaling pathway. A, nuclear proteins were isolated from THP-1 cells after mock (Lane 1) or MIP-1{alpha} (Lanes 2–7) stimulation. The labels on Lanes 3–7 indicate the pretreatment for the cells. Analysis was carried out as detailed in the legend to Fig. 1Citation . B, control experiment for A. The concentrations of each reagent used are specified in "Materials and Methods." Staur., staurosporine; bis. 1, bisindolylmaleimide 1; Forsk., forskolin.

 
Because CRE is a cAMP-responsive target sequence, we tested the effects of Rp-cAMP on forskolin, a known inducer of the adenyl cyclase pathway (26 , 47) . The results shown in Fig. 3Citation (B-II) revealed that Rp-cAMP can block forskolin-induced, PKA-dependent binding to CRE. This experiment correlates with the results from Fig. 3Citation (A-II) in which Rp-cAMP failed to inhibit MIP-1{alpha}-induced CRE activation. TPA, a well-known stimulator of PKC (26 , 48) , was used as a control for the MIP-1{alpha}-induced AP-1 activity. Staurosporine (10-7 M) inhibited the TPA-stimulated, PKC-dependent AP-1 activity (Fig. 3Citation , B-I). Because staurosporine has been shown to potentially inhibit other serine-threonine protein kinases, the inhibited AP-1 or CRE activity shown in Lanes 5 and 6 of Fig. 3Citation (A-I or A-II) could result from the combined effect of PKC and other protein kinase pathways. To that end, we used a highly selective PKC inhibitor, bisindolylmaleimide 1 (49 , 50) , to treat the THP-1 cells. As shown in Fig. 3Citation (Lane 7 in panels A-I and A-II), bisindolylmaleimide 1 significantly inhibited the AP-1 activity induced by MIP-1{alpha} but had little effect on CRE activity. By combining the results in Fig. 3Citation , it appears that MIP-1{alpha} induction of AP-1 activity is PKC dependent, whereas its induction of CRE activity is PKA independent with minimal involvement of PKC. MIP-1{alpha} appears to induce CRE activation through a different pathway than that of forskolin, one that is independent of PKC.

Characterization of the Constituents in the MIP-1{alpha}-induced AP-1 and CRE Complexes.
To identify the constituents of the MIP-1{alpha}-induced AP-1 and CRE complexes, supershift assays were performed. Antibody to the jun family of transcription factors (including c-jun, jun B, and jun D) supershifted not only the AP-1 binding complex induced by MIP-1{alpha} but also the CRE binding complex (Fig. 4)Citation . Anti-fos family (including c-fos, fos B, fra-1, and fra-2) antibody further shifted the AP-1 complex, indicating that fos family members may be involved in the MIP-1{alpha}-induced AP-1 complex. Antibody to CREB-1 and CREB-2 transcription factors did not cause a supershift of the MIP-1{alpha}-induced CRE complex, whereas another member of the CREB/ATF family, ATF-2, did appear in this CRE complex. The observation that CREB family members were not present in the CRE-protein complex likely explains why the cAMP pathway inhibitor, Rp-cAMP, did not abrogate MIP-1{alpha}-induced CRE binding activity. Moreover, the presence of jun acting as a common component in both the AP-1 and CRE complexes induced by MIP-1{alpha} may explain the similarity in the AP-1 and CRE binding patterns (Fig. 1Citation , B-I and B-II, C-I and C-II).



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Fig. 4. The nuclear protein components that bind AP-1 and CRE sequences. Nuclear proteins from MIP-1{alpha}-treated THP-1 cells were interacted with AP-1 (A) or CRE (B) target sequences for 10 min before they reacted with antibodies against the leucine zipper family of transcription factors. Lane 1, binding activity of MIP-1{alpha}-treated THP-1 cells without the addition of antibody. Lanes 2–7, reactions with added antibodies. Arrows, DNA-protein-antibody supershift complex. C, a Western blot using the probes of antibodies to c-Jun (C-I) and Jun D (C-II). The labels show the time points for protein extraction after MIP-1{alpha} treatment. Single arrow, Jun D protein expression; double arrow, high molecular weight, nonspecific bands that react with the anti-c-Jun antibody.

 
The results of serial Western blots demonstrated that Jun D was the Jun family member detected in both the AP-1 and CRE complexes induced by MIP-1{alpha}. c-Jun (Fig. 4Citation , C-I) was not expressed in THP-1 cells, either in the presence or absence of MIP-1{alpha}, and Jun B antibody did not probe any band in the total protein of the THP-1 cells (data not shown). The data in Fig. 4Citation (panel C-II) indicate that THP-1 cells expressed Jun D constantly (Lane 1), and this expression did not change 1, 12, and 24 h (Lanes 2, 3, and 4) after MIP-1{alpha} stimulation. These results indicated that Jun D is the only member of the Jun family expressed in the THP-1 cells. This implies that Jun D is present in the AP-1 binding complex.

Binding to AP-1 and CRE Target Sequences Is Induced by RANTES and IL-8 in THP-1 Cells.
To further investigate the signaling involved in MIP-1{alpha}-induced AP-1/CRE activation, the closely related ß-chemokines MIP-1ß and RANTES, and the unrelated {alpha}-chemokine IL-8, were studied in the model THP-1 myeloid cell line. As shown in Fig. 5Citation , THP-1 cells treated with MIP-1ß failed to show AP-1 or CRE activity (Lane 4), whereas cells stimulated by RANTES induced the activation of both AP-1 and CRE (Lane 5). Interestingly, IL-8, which binds to a different set of receptors (e.g., CXCR1 and CXCR2), also induced AP-1/CRE binding activity (Lane 8). Lanes 6 and 9 show that an unlabeled AP-1 or CRE oligo can compete binding to the labeled target sequence, and Lanes 7 and 10 show that the unlabeled mutant AP-1 or CRE sequence does not compete with the labeled probes. To determine whether the pathway by which {alpha}-chemokines induced AP-1 and CRE activation is different from that of ß-chemokines, we used the protein kinase A inhibitor Rp-cAMP and the PKC inhibitor staurosporine. The studies indicated that the PKA inhibitor as well as the PKC inhibitor blocked IL-8-induced AP-1 and CRE activity (data not shown). Thus, IL-8 induces AP-1 and CRE activation through a different pathway than that of MIP-1{alpha}.



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Fig. 5. AP-1 and CRE activity are induced by RANTES and IL-8. Nuclear proteins were isolated from mock-, MIP-1{alpha}-, MIP-1ß-, RANTES- and IL-8-treated THP-1 cells and analyzed as described in the legend to Fig. 1Citation .

 
JNK and ERK Activation Are Mediated by ß-Chemokines.
Chemokine coupled G-proteins (Gi and Gq) are also known to activate various MAP kinase pathways (51, 52, 53, 54) . JNK, a member of the MAP kinase family, phosphorylates c-jun at Ser-73 and Ser-63, both located within the transactivation domain of c-jun. Phosphorylation of these residues, which does not affect DNA binding activities, potentiates the ability of c-jun to activate transcription (55) . As an independent approach to elucidate the role of jun in chemokine signaling pathways, we evaluated the correlation between chemokine stimulation and JNK activation in THP-1 cells. THP-1 cells were treated with 30 nM MIP-1{alpha} or IL-8; total cellular proteins were isolated 5, 15, or 30 min after these treatments, and the assay for JNK activity was performed. As shown in Fig. 6Citation (A-I), within 5 min after stimulation by MIP-1{alpha}, a 3–4-fold increase in JNK activation was observed. This activity gradually returned to baseline by 30 min. IL-8 had a similar effect but was somewhat weaker in inducing JNK activation as compared with MIP-1{alpha}. The MIP-1{alpha}-induced activation of the other MAP kinases, ERK-1/ERK-2 and p38 MAP kinase, is shown in Fig. 6BCitation . ERK-1/ERK-2 activities were increased 4–5-fold by MIP-1{alpha} treatment, with a peak at 15 min (Fig. 6Citation B-II, Lanes 1, 2, and 3). The activity of p38 MAP kinase did not increase for up to 30 min after MIP-1{alpha} stimulation (Fig. 6Citation B-II, Lanes 4, 5, and 6).



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Fig. 6. Kinase assays for MIP-1{alpha}- and IL-8-treated cells. A-I and B-I, the increase in kinase activation is plotted against the time when proteins were isolated after the indicated chemokine treatments. Kinase activity was measured by densitometry, and the values on the Y axes reflect the fold of kinase activity induced by chemokines versus the basal activity (kinase activity before chemokine treatment). The means are derived from three independent experiments; bars, SD. A-II, a gel representative of the three JNK assays. Lane 1, JNK activity in control THP-1 cells. Lanes 2–4, cells treated by MIP-1{alpha}. Lanes 5–7, cells treated by IL-8. B-II, the gels represent the ERK and p38 MAP kinase assays after MIP-1{alpha} treatment. Lanes 1–3, ERK assay. Lanes 4–6, p38 MAP kinase assay.

 
Alteration of AP-1 and CRE Binding Activity by Exogenous Chemokines from HIV-infected THP-1 Cells.
Myeloid cells of monocytic origin are susceptible to HIV-1 infection, and retrovirus infection is known to alter cell responses and signaling (56 , 57) . We investigated the effects of HIV on the chemokine-induced activation of transcription factors. As shown in Fig. 7Citation , the basal levels of AP-1 and CRE activation were markedly increased in HIV-infected THP-1 cells compared with uninfected cells (Fig. 7ACitation , Lanes 1 and 2). Thirty nM MIP-1{alpha} treatment did not induce a further binding activity of either the AP-1 or the CRE target sequence in the HIV-1 infected cells (Lane 3), although when a higher dose of MIP-1{alpha} (100 nM) was added, the binding activities of AP-1 and CRE were slightly increased (Lane 4).



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Fig. 7. A, AP-1 and CRE are constantly activated in HIV-infected THP-1 cells. Nuclear proteins were isolated from noninfected (Lane 1) and HIV-infected (Lane 2) THP-1 cells and analyzed as described in the legend to Fig. 1Citation . Lane 5, HIV-infected THP-1 cells treated by anti-MIP-1{alpha} antibody for 4 h. B, HIV gp120 induces AP-1 activation in THP-1 cells. Nuclear proteins were isolated from mock-treated (Lane 1) and gp120-treated (Lane 2) THP-1 cells and analyzed as described in the legend to Fig. 1Citation . C, HIV-infected THP-1 cells express high levels of MIP-1{alpha}. C-I, Northern blot analysis. Total RNA was prepared from noninfected (Lane 1) or HIV-1 IIIB-infected (Lane 2) THP-1 cells followed by separation on 1% agarose gel and transfer to nylon membrane. The blot was hybridized with the MIP-1{alpha} probe (for details, see "Materials and Methods"). Arrow, MIP-1{alpha} mRNA. C-II, total RNA in Lanes 1 and 2 of C-I was stained with ethidium bromide as a control for the loading amounts. The two bands are the 28S and 18S rRNA. C-III, ELISA. Supernatants were prepared from mock-treated ({blacksquare}), LPS-treated ({square}), or HIV-1 IIIB-infected () THP-1 cells. The quantification of MIP-1{alpha} in the supernatants was determined by ELISA, as described in "Materials and Methods." The quantity of MIP-1{alpha} shown on the Y axis represents the amount produced by 106 cells/ml over 24 h. The means are from triplicates of three independent experiments; bars, SD.

 
To determine the nature of the signals involved in the activation of AP-1 and CRE upon HIV infection, several possible mechanisms were investigated. HIV envelope glycoproteins have been reported to induce the activation of AP-1 in CD4+ T cells (58) . We therefore studied whether treatment of THP-1 cells with recombinant HIV-1 IIIB gp120 would induce AP-1/CRE binding activation. As shown in Fig. 7BCitation , binding to the transcriptional element AP-1 was induced by the HIV envelope in the uninfected myeloid cells (B-I), whereas there was no detectable change in binding activity to the CRE sequence when stimulated by gp120 (B-II). This finding could account for the altered constitutive binding activity of the AP-1 sequence in uninfected myeloid cells after their contact with HIV gp120 released by neighboring HIV-infected cells.

Furthermore, because HIV-infected cells may secrete various cytokines and chemokines, we considered whether autocrine or paracrine release of such soluble mediators could cause the sustained activation of transcription factors. Northern blot analysis was performed to detect MIP-1{alpha} expression in THP-1 cells before and after HIV-1 IIIB infection. A dramatic increase in MIP-1{alpha} mRNA expression was observed in HIV-infected cells compared with uninfected cells (Fig. 7Citation , C-I and C-II). IL-8 mRNA expression showed an increase similar to MIP-1{alpha} expression after HIV-1 infection (data not shown). To confirm that the ß-chemokine was also expressed at the protein level in HIV-infected THP-1 cells, ELISA was used to quantitate MIP-1{alpha} secretion from THP-1 cells before and after HIV-1 IIIB infection. Fig. 7Citation , C-III shows that THP-1 cells constitutively express little MIP-1{alpha}. However, after HIV infection, these cells showed a significant increase in MIP-1{alpha} secretion, at a level similar to cells stimulated by fetal LPS, a known inducer of ß-chemokine secretion in leukocytes. These results suggest an autocrine or paracrine mechanism for the sustained transcriptional activation observed in HIV-1-infected myeloid cells. In contrast, when HIV-infected THP-1 cells were treated with antibody against MIP-1{alpha}, the binding activity to both the AP-1 and CRE sequences was decreased significantly (Fig. 7ACitation , Lane 5).


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have characterized transcriptional activation by ß-chemokines in human monocytic cells and observed that the AP-1 element is modulated via a PKC-dependent mechanism. On the basis of prior information on pathways of transcriptional regulation stimulated by various inducers in other cell types, it would be expected that an inhibitor of the PKC pathway would block AP-1 activation (25 , 59, 60, 61) . Our observation that an inhibitor of PKA did not block CRE binding activity is interesting. This finding suggests that ß-chemokine regulation of CRE binding activity may not be via the classical PKA and CREB pathway. The additional observation that a jun family member and ATF-2 were in the CRE-protein complex provides a likely explanation for this phenomenon. Binding of jun-jun homodimers or jun-ATF-2 heterodimers to the CRE elements has been reported (62, 63, 64) . Our previous work also demonstrated certain cross-family binding to the closely related target sequence, the UV-responsive element (42 , 65) . Such cross-talk could be operative in ß-chemokine-induced activation of CRE as well.

C-C chemokine receptors are known to be coupled to Gi proteins (2 , 13 , 66) . The activation of Gi protein would be expected to inhibit cAMP turnover and consequently down-regulate PKA, which could decrease the binding of CREB to the CRE target sequence. In our studies, the PKA inhibitor Rp-cAMP did not block CRE binding activity in THP-1 cells, indicating that PKA is not involved in this ß-chemokine-induced activation. In contrast, 10-7 M staurosporine blocked the CRE activation induced by MIP-1{alpha}. Because the highly selective PKC inhibitor, bisindolylmaleimide 1, did not inhibit CRE activity, the staurosporine-induced inhibition of this activity could result from its nonspecific inhibition of other serine/threonine kinases, such as MAP kinases. ß-Chemokine activation of Gi protein has been reported to activate the MAP kinase pathway. One member of the MAP kinase family, JNK, can phosphorylate and activate both jun and ATF-2 and was activated by MIP-1{alpha} in our system. Taken together, these data suggest that MIP-1{alpha}-induced CRE activation is not through the PKA pathway but rather through the MAP kinase pathway.

We extended our studies on MIP-1{alpha} and found induction of AP-1 and CRE by the related ß-chemokine RANTES as well as by the unrelated {alpha}-chemokine IL-8. Two highly homologous seven-transmembrane domain receptors for IL-8, CXCR1 and CXCR2, have been identified and are reported to be coupled to the G proteins, Gi and Gq (67 , 68) . The activation of Gi protein would be expected to down-regulate the PKA pathway, but our results showed that PKA participated in AP-1 and CRE activation. This finding suggests that a certain type of Ca2+-stimulated adenyl cyclase, which is not inhibited by Gi coupled receptors (69) , may play a role in this pathway. It is also possible that, in monocytes, IL-8 receptors are coupled to different G proteins than those reported in neutrophils. Furthermore, IL-8 stimulates the Ras/Raf/MAP kinase pathway (70) , and Jun and Fos are transcription factors known to be downstream of these kinases. Thus, it is likely that multiple pathways are involved in IL-8-mediated transcriptional activation. Our data indicate that although common transcriptional elements may be involved in chemokine effects, different signaling pathways may be used.

The results from the Western blot help to elucidate the mechanism for AP-1 binding. The increased AP-1 binding activity induced by MIP-1{alpha} could result from increased gene expression, posttranslational modification, or from protein accumulation. Induced gene expression at the protein level involves de novo protein synthesis and may require a 12–24-h time period (25) . Because the increased binding activity in the gel shift reflects induction of gene expression at the protein level, rather than at the mRNA level, this induction may not explain the increased binding that was observed within 30 min. If the mechanism is by posttranslational modification, such as protein phosphorylation, it could happen within minutes, and the amount of protein would not necessarily be changed. Because increased amounts of Jun D protein were not detected after MIP-1{alpha} treatment, activation of Jun D may indeed be by posttranslational modification. However, if MIP-1{alpha} induces Fos family member or ATF-2 accumulation, it could happen in a short period of time and contribute to the increased binding activity of AP-1 or CRE.

The observation that MIP-1{alpha} induced the binding of AP-1 and CRE sequences within 30 min suggests a mechanism involving posttranslational modification or accumulation of preexisting proteins. The decrease in binding activity appeared 1.5–2 h after MIP-1{alpha} treatment. This time period correlates with certain chemokine-regulated functional responses, such as monocyte chemotaxis. There are abundant reports demonstrating that activation of AP-1 or CRE will induce expression of a variety of cytokines or chemokines. Activation of AP-1 or CRE/AP-1-like elements could result in the induction of IL-8, MCP-1, or RANTES expression (71, 72, 73) . Thus, MIP-1{alpha} stimulation of AP-1 and CRE activation may play a role in the expression of multiple chemokines in response to stimulation by the bacterial product LPS or during inflammatory responses to other agents.

Interestingly, HIV-1 infection of THP-1 cells resulted in the constitutive activation of binding to both AP-1 and CRE motifs. We considered several mechanisms for this phenomenon. HIV-1 envelopes interacting with the chemokine receptor CXCR4 or CCR5 could initiate multiple signaling pathways (74) . Chirmule et al. (58) reported that in T cells, native envelope glycoproteins of HIV could induce activation of the transcription factor AP-1; this effect was mediated through the surface CD4 molecule. In cultures of HIV-infected THP-1 cells, where some cells are productively infected and neighboring cells uninfected (similar to the monocyte-macrophage population in various tissues), we found activation of AP-1 and CRE. This activation may result from the HIV envelope glycoprotein gp120 inducing transcriptional activation in myeloid cells, as well as from induction of MIP-1{alpha} secretion by the retrovirus. Our findings indicate that physiological transcriptional responses of monocyte-macrophages to chemokines may be blunted by HIV infection and thus contribute to impairment in this cell function in patients with AIDS.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines.
The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Bethesda, MD), found to be Mycoplasma free, and maintained in DMEM supplemented with 10% fetal bovine serum. Cells were grown at 37°C in 5% CO2.

HIV Infection.
Preparation of titered stocks of HIV-1 IIIB and HIV infection of THP-1 cells were performed as described previously (75) . In brief, THP-1 cells were challenged with 0.25 ml of HIV-1 IIIB (1–2 x 105 cpm/ml by reverse transcriptase assay) for 4 h. HIV-1 viral supernatants were then removed, and the cells were washed three times with HBSS and maintained in 1 ml of growth medium. These THP-1 cells were confirmed to be infected based on reverse transcriptase assay of culture supernatants and used in the experiments detailed below.

The recombinant HIV envelope glycoprotein (gp120) of the HIV-1 IIIB isolate produced in a baculovirus expression system was purchased from American BioTechnologies, Inc. (Cambridge, MA). The THP-1 cells were treated with 50 nM gp120 for 1 h at 37°C in serum-free DMEM.

Reagents.
The chemokines MIP-1{alpha}, MIP-1ß, RANTES, and IL-8 were obtained from R&D Systems, Inc. (Minneapolis, MN). Unless specified otherwise, the THP-1 cells were treated with 30 nM of each chemokine. TPA, forskolin, Rp-cAMP, and staurosporine were purchased from Sigma Chemical Co. (St. Louis, MO). Bisindolylmaleimide 1 was purchased from Calbiochem (San Diego, CA) and dissolved in DMSO. TPA (100 ng/ml), forskolin (20 µM), staurosporine (10-7 and 10-9 M), or bisindolylmaleimide 1 (50 nM) was added to cells 4 h before and Rp-cAMP (10 and 50 nM) was added 1 h before cell lysis and protein preparation.

Antibodies.
The antibodies used in the Western blotting and supershift assays included antibodies to all Jun family members, to all Fos family members, and other specific antibodies: c-Jun, Jun B, Jun D, CREB-1, CREB-2, ATF-1, and ATF-2, as well as the antibodies to JNK, ERK-1, ERK-2, and p38 MAP kinase. All of these antibodies were obtained commercially (Santa Cruz Biotechnology, Inc., Santa Cruz Biotechnology, CA). The antibody anti-MIP-1{alpha} was a generous gift from LeukoSite, Inc. (Cambridge, MA).

Isolation of Nuclear Proteins.
Cells were serum-starved in a 37°C incubator for 4 h, followed by 30 min of mock or chemokine treatment. Nuclear proteins were then isolated using standard methods (76, 77, 78, 79, 80) . The protease inhibitors antipain (1 µg/ml), leupeptin (1 µg/ml), phenylmethylsulfonyl fluoride (0.1 mM), and the phosphatase inhibitor Na3VO4 (1 mM) were added to all buffers except the dialysis buffer. DTT (1 mM) was added to all buffers. After dialysis, the protein concentrations were determined using an assay kit (Bio-Rad Laboratory, Hercules, CA), and aliquots of the proteins were stored at -80°C.

EMSA.
Double-stranded oligonucleotides containing the consensus binding site for AP-1 (5'-CGC TTG ATG ACT CAG CCG GAA-3'), mutated AP-1 (5'-CGC TTG ATG ACT TGG CCG GAA-3'), or CRE (5'-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3'), mutated CRE (AGA GAT TGC CTG TGG TCA GAG AGC TAG-3') were purchased commercially (Santa Cruz Biotechnology). An oligonucleotide representing a dimer of the UV-responsive element sequence (ACT ATG ACA ACA GCT ATG ACA ACA GT; Refs. 40, 41, 42 ) was synthesized by Integrated DNA Technology, Inc. (Coralville, IA). All oligonucleotides were labeled with [{gamma}-32P]ATP (3000 Ci/mmol; DuPont NEN, Boston, MA) using polynucleotide kinase (Promega Corp., Madison, WI) according to standard procedures. The labeled DNA (0.4 ng; 4400 cpm) was incubated with 5 µg of nuclear proteins (as specified in "Results") for 10 min at room temperature, in the presence of 100 ng of poly(deoxyinosinic-deoxycytidylic acid) oligomer (Boehringer Mannheim, Indianapolis, IN) and DNA-binding buffer as described previously (41) . The complexes were then separated on a 7.5% polyacrylamide gel and autoradiographed. The results shown in each of the figures are representative of findings from at least three independent experiments.

In supershift reactions, 10 µg of nuclear proteins were incubated with a radiolabeled probe. After 10 min, specific antibodies were added to the DNA binding reaction mixtures and incubated for another 2 h or overnight at 4°C. The reaction mixtures were then separated on a 4% polyacrylamide gel containing 0.7% glycerol.

Transfection and CAT Assays.
The CAT assays were performed using a standard method as described previously (42) . Twenty µg of 5x AP-1-CAT plasmid (14) , 3x CRE-CAT plasmid, or the parent pCAT-basic plasmid were electroporated together with a RSV-ß-gal construct (10 µg) into 107 cells using 230 V, 960 mF in 1x HEPES buffer. After MIP-1{alpha} treatment, total proteins were collected. Levels of CAT activity were determined by TLC, and the conversion of chloramphenicol to the acetylated form was quantitated by densitometry. Correction for transcriptional activity was based on values obtained from control (without MIP-1{alpha} treatment) AP-1-CAT or CRE-CAT activities. The transcription efficiency was normalized against ß-gal activities that were measured in the same protein preparations. The results represent three independent experiments with triplicates for each dose.

Western Blot Analysis.
Western blot analysis was performed as described previously (26) . Briefly, total proteins were prepared from the THP-1 cells receiving sham treatment or after 1, 6, and 24 h MIP-1{alpha} (30 nM) stimulation. Fifty µg of the total proteins were boiled for 5 min in the presence of Laemmli sample buffer and separated by 10% SDS-PAGE. The gels were then transferred to nitrocellulose membranes. The anti-jun family antibody was diluted to a concentration of 1:400 before use. Immunoreactive bands were detected with an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech).

Kinase Assay.
Cell lysates were immunoprecipitated with anti-JNK, anti-ERK-1, anti-ERK-2, or anti-p38 MAP kinase antibodies (Santa Cruz Biotechnology, Inc.), and rabbit IgG was used as a negative control. The immune complexes were washed twice with RIPA buffer and once in kinase buffer (81, 82, 83) . The complex was then incubated in 20 µl of kinase buffer for 30 min at room temperature. The kinase buffer contained 50 ng/µl of recombinant GST c-Jun (1–79 amino acids; Santa Cruz Biotechnology, Inc.) as JNK substrate, 7 mg of myelin basic protein each (Santa Cruz Biotechnology, Inc.) as ERK and p38 MAP kinase substrates, and 5 µCi of [{gamma}-32P]ATP (3000 Ci/mmol; DuPont NEN). The reaction was terminated by adding 2x SDS sample buffer and boiling the sample for 5 min. Proteins were separated by 12% SDS-PAGE and detected by autoradiography. The kinase activity of each band on the films was quantified by densitometry.

Northern Blot Analysis.
RNA extraction, electrophoresis, gel transfer to nylon membranes, and blot hybridization were performed by standard procedures. Blots were washed twice at 65°C in 2x SSC and 0.1% SDS for 15 min. Probes were labeled using incorporation of digoxigenin-11-dUTP (Boehringer Mannheim) during routine PCR. The sequences for MIP-1{alpha} primers are 5'-CGC CTG CTG CTT CAG CTA CAC-3' and 5'-TGT GGA GGT CAC ACG CAT GTT-3' with a product probe of 280 bp; the sequences for IL-8 primers are 5'-CGA TGT CAG TGC ATA AAG ACA-3' and 5'-TGA ATT CTC AGC CCT CTT CAA AAA-3' with a product probe of 225 bp (84) . Detection, followed by autoradiography, was performed by using chemiluminescence with anti-digoxigenin-AP antibody, which conjugates to alkaline phosphatase, DIG-Wash, Block Buffer Sit, and the CDP-Star [25 mM disodium 4-chloro-3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phosphate], a chemiluminescent alkaline phosphatase substrate (Boehringer Mannheim). The detection procedure followed the recommendations of the manufacturer. The amount of RNA loaded was monitored using 28S and 18S rRNA stained with ethidium bromide.

ELISA.
HIV-infected THP-1 cells or noninfected THP-1 cells (106/ml) were cultured in fresh medium for 24 h; cells treated with LPS (Sigma Chemical Co.) were cultured in serum-free DMEM with 2 µg/ml LPS for 24 h. MIP-1{alpha} concentrations in the cell culture supernatant were then detected by the Human MIP-1{alpha} Quantikine kit (R&D Systems, Inc.). The assay procedure followed the recommendations of the manufacturer. Absorbency was determined using a microtiter plate reader set to 450 nM. A standard curve was generated for each set of samples, which were assayed to calibrate the concentrations of MIP-1{alpha}.


    Acknowledgments
 
We thank Drs. Ramesh Ganju and Gita Venkatakrishnan of Beth Israel Deaconess Medical Center for helpful comments and Dr. Ruth Halabanin from Yale University School of Medicine for offering the CRE-CAT construct. We thank Janet Delahanty for editorial assistance and preparation of figures. We also thank Evelyn Gould for assistance with the figures and Simone Jadusingh for typing assistance.


    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 Supported by NIH Grants HL 55187, HL 53745, and HL 43510 and grants from the David Geffen Foundation and the Robert Farber Foundation. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, Division of Experimental Medicine, Harvard Institutes of Medicine-BIDMC, 4 Blackfan Circle, Third Floor, Boston, MA 02115. Phone: (617) 667-0070; Fax: (617) 975-5244. Back

4 The abbreviations used are: MIP, macrophage inflammatory protein; IL, interleukin; AP-1, activation protein-1; CRE, cyclic AMP responsive element; CREB, CRE binding protein; RANTES, regulated on activation, normal T cell expressed and secreted; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA responsive element; ATF, activating transcription factor; MCP, monocyte chemoattractant protein; PKA, protein kinase A; PKC, protein kinase C; EMSA, electrophoretic mobility shift assay; LPS, lipopolysaccharide; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; URE, UV-responsive element; CAT, chloramphenicol acetyltransferase; ß-gal, ß-galactosidase. Back

Received for publication 8/30/00. Revision received 3/ 1/01. Accepted for publication 3/ 1/01.


    References
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 Discussion
 Materials and Methods
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