Regulation of Nuclear Factor-κB by Corticotropin-Releasing Hormone in Mouse Thymocytes

Abstract

CRH, a major mediator of the stress response, has been shown to exert potent immunomodulatory effects in vivo, through mechanisms that have not been elucidated yet. To determine the molecular pathways mediating the proinflammatory effects of peripheral CRH, we studied its role in the activation of nuclear factor-κB (NF-κB), a transcription factor crucial for the regulation of a variety of inflammatory mediator genes. Our studies demonstrate that, in mouse thymocytes, CRH induces the NF-κB DNA-binding activity in a time- and dose-dependent manner, with parallel degradation of its inhibitor protein inhibitor of NF-κB. The effect of CRH is not inhibited by dexamethasone and is mediated by the protein kinase A and protein kinase C signaling pathways. In vivo, we show that CRH-deficient mice respond to lipopolysaccharide administration by reduced activation of thymus NF-κB, despite their significantly elevated proinflammatory cytokine and their low corticosterone levels. These findings suggest a putative molecular pathway mediating the proinflammatory effects of peripheral CRH through induction of the NF-κB DNA binding activity.

CRH, A 41-AMINO-ACID peptide originally identified by its ability to stimulate pituitary ACTH secretion (1), is a key integrator of the neuroendocrine response to stress. In addition to its central role in activating the hypothalamic-pituitary-adrenal axis (2), CRH also directly participates in the inflammatory response, as a locally expressed proinflammatory factor (3–5). CRH and its receptor(s) are expressed in mouse and human leukocytes and immune organs (6–11). Peripheral CRH has been identified in inflamed rodent (3, 12, 13) and human tissues from different inflammatory diseases, such as rheumatoid arthritis and osteoarthritis (14) and inflammatory bowel disease (15). Peripheral sensory afferent type C fibers and postganglionic sympathetic nerves also express CRH (16, 17) and have been postulated as another source of the immune CRH (18). Other studies have shown antiinflammatory effects of CRH and its related peptides after their local administration in experimental inflammatory models (19–23). In vitro immunomodulatory properties of CRH include stimulation of T and B lymphocyte proliferation (24), induction of proinflammatory cytokine secretion (25), IL-2 receptor expression (26), acute-phase protein expression (27), natural killer cell-mediated lysis (28), as well as mast cell degranulation (29). In addition, CRH stimulates the expression of proopiomelanocortin-derived peptides in leukocytes (30), suggested to participate in inflammation-induced analgesia (12, 31). Despite the increasing evidence for direct immunomodulatory actions of CRH, the molecular mechanisms mediating these effects have not been elucidated yet.

Nuclear factor-κ B (NF-κB), a transcription factor regulating the expression of a variety of proinflammatory genes, is a heterodimer composed mainly by the Rel A (p65) and NF-κB1 (p50) subunits. In inactive states NF-κB is sequestered in the cytoplasm by its inhibitor protein, IκBα (32, 33). Extracellular stimuli cause phosphorylation and subsequent degradation of IκB, which allows translocation of NF-κB into the nucleus where it binds to specific promoter sites of its target genes and induces their transcription, including IκBα itself (34). NF-κB DNA-binding activity can be induced by a variety of immune system activators including mitogens, cytokines, endotoxin, viral proteins, and nitric oxide (35, 36). Activated NF-κB stimulates proinflammatory mediators such as cytokines, cell adhesion molecules, class I and II major histocompatibility antigen, complement factors, and acute phase response proteins by binding specific regions of their promoters (36, 37). Increasing evidence supports direct involvement of NF-κB in the progress of several diseases such as inflammatory bowel disease and rheumatoid arthritis (38–41). The inability of NF-κB-deficient animals to generate a normal immune response after various stimuli (42, 43), as well as the ability of specific NF-κB inhibitors to block the development of several inflammatory models (44–46), provide more evidence on the significance of NF-κB for the inflammatory process. In this study, we have tested the hypothesis in vitro and in vivo (47) that CRH induces the NF-κB DNA binding activity in mouse thymocytes, as a putative pathway of the previously shown proinflammatory effects of CRH in rodents (3, 4, 13).

RESULTS

CRH-Induced NF-κB Activation in Mouse Thymocytes

We used primary cultures of mouse thymocytes obtained from female adult mice. The time-course of NF-κB activation after CRH (10⁻⁷m) treatment from 0 up to 3 h was examined by EMSA. As shown in Fig. 1A, CRH induced a time-dependent up-regulation of NF-κB DNA-binding activity that appeared as early as 10 min after CRH treatment (lane 2), peaked between 30 and 60 min (lanes 3 and 4), and had declined substantially 3 h after treatment (lane 5). Time-dependent up-regulation of the NF-κB DNA-binding activity was also induced by lipopolysaccharide (LPS; 5 μg/ml; lanes 6–8), used as a positive control (48). Lane 1 in Fig. 1A depicts the constitutive NF-κB DNA binding activity, evident in untreated cells as previously reported (49). Abolishment of the NF-κB DNA-binding complex in the presence of excess amount of unlabeled NF-κB oligonucleotide (competitor; lane 9) confirms the specificity of the binding reaction. The degree of NF-κB activation 1 h after treatment with CRH was dose dependent with plateau effect at doses 10⁻⁷m (Fig. 1B) and higher (data not shown).

Fig. 1.

Induction of NF-κB DNA Binding Activity by CRH in Mouse Thymocytes EMSA analysis of nuclear extracts from mouse thymocytes treated with CRH, LPS, or nothing (control). A, Time-course of NF-κB activation by CRH. CRH (10⁻⁷m) treatment for the indicated times (lanes 2–5) induced a time-dependent increase of NF-κB activation as did by LPS (lanes 6–8). Lane 1, Control; lane 9, CRH-treated cells incubated with excess amount of unlabeled NF-κB oligonucleotide (competitor). B, NF-κB activation 1 h after treatment with graded doses of CRH (lanes 1–3). Lane 4, Control; lane 5, CRH-treated cells incubated with competitor. Results represent one of three individual experiments. C, Effect of α-helical CRH (10⁻⁶m; lane 4), and unlabeled NF-κB oligonucleotide competitor (lane 6), or unlabeled mutant NF-κB oligonucleotide competitor (lane 5) on the CRH-induced activation of NF-κB (lane 3). D, Quantitative analysis of the effect of CRH on NF-κB complex from three representative experiments. The density of the specific band was measured using NIH imaging software. Data are presented as mean ± sem from the means of triplicates for every experimental condition from each experiment. **, P ≤ 0.01 compared with control. In all panels, n.s. depicts a nonspecific band shown as an indicator of loading.

Blockade of the CRH-induced NF-κB activation by cotreatment with the peptide CRH antagonist, α-helical CRH 9–41 (50) [Fig. 1, C (lanes 3 and 4) and D (lane 4)], confirmed the specificity of the effect. The specificity of the binding interaction was further confirmed by the lack of an effect of the mutant NF-κB oligonucleotide competitor (Fig. 1C, lane 5). Figure 1D depicts quantitative analysis of the LPS, CRH, and α-helical CRH effect on NF-κB DNA binding activity from three experiments.

Dexamethasone Effect on the CRF-Induced NF-κB Activation

Glucocorticoid down-regulates CRH expression in most of the sites the latter is produced, whereas it inhibits the activation of NF-κB induced by several inflammatory stimuli (51–53). To assess the effect of glucocorticoid on the CRH-induced NF-κB activity, we pretreated mouse thymocytes with dexamethasone (10⁻⁶m) for 2 h before addition of either CRH (10⁻⁷m) or LPS. As shown (Fig. 2, A and C), dexamethasone had no effect on the constitutive activation of NF-κB (lanes 1 and 2), whereas it inhibited the CRH- (lanes 5 and 6) as well as the LPS-induced NF-κB DNA-binding activity (lanes 3 and 4).

Fig. 2.

Effect of Dexamethasone (Dex) (A) and Protein Kinase Inhibitors (B) on the CRH-Induced NF-κB Activation as Determined by EMSA Analysis A, Mouse thymocytes were pretereated with dexamethasone or vehicle for 2 h before addition of CRH, LPS, or vehicle. Results represent one of three individual experiments. B, Mouse thymocytes were treated with either the PKC inhibitor H7 (10⁻⁴m), or the PKA inhibitor H89 (10⁻⁴m) for 30 min before stimulation for 1 h with CRH (10⁻⁷m). Results represent one of four individual experiments. C, Quantitative representation of the experimental treatments shown in panels A and B, from three individual experiments represented as percent changes over control values. The density of the specific band was measured using NIH imaging software and was normalized for the density of the nonspecific band depicted at the bottom of each gel shift. Data are presented as mean ± sem from three different samples for every treatment condition. *, P ≤ 0.01 compared with control, #, P ≤ 0.01 from LPS-treated cells, @, P ≤ 0.01 from CRH-treated cells. n.s. Nonspecific band shown as an indicator of loading.

Role of Protein Kinase A (PKA) and Protein Kinase C (PKC) on the CRH-Induced NF-κB Activation

PKA and PKC mediate the effects of CRH after binding to its receptor(s) (54, 55), whereas they have been also implicated in the signal transduction pathways leading to NF-κB activation (56, 57). To define the role, if any, of these kinases in the CRH-induced NF-κB activation, EMSA was performed in nuclear extracts of mouse thymocytes treated with either the PKA inhibitor H89 (10⁻⁴m) or the PKC inhibitor H7 (10⁻⁴m) 30 min before CRH (10⁻⁷m) administration. As shown in Fig. 2B, H89 resulted in a slight inhibition of the constitutive NF-κB DNA binding activity, whereas it abolished the CRH effect (lanes 4 and 6). Interestingly, H7 increased both the constitutive as well as the CRH-induced NF-κB DNA binding activity (lanes 3 and 5).

Figure 2C illustrates quantitative analysis of the data obtained from three individual experiments on the effect of the treatments shown above (Fig. 2, A and B) on the NF-κB DNA binding activity in mouse thymocytes. All changes are expressed as percent change over the constitutive (control) NF-κB activity.

Effect of CRH on IκBα Protein Expression in Mouse Thymocytes

NF-κB activation has been associated to phosphorylation and subsequent degradation of IκBα. We evaluated whether the effect of CRH on NF-κB activation is related to changes in the abundance of IκBα by Western blot analysis of CRH (10⁻⁷m)-treated thymocytes. Time-dependent reduction of IκBα protein expression with a marked decrease 30 min after CRH addition is shown in Fig. 3A. The CRH effect was blocked by coaddition of α-helical CRH (Fig. 3B, lane 2) in analogy to its effect on the NF-κB activation described above. Figure 3C represents the quantitative analysis of three individual experiments of the effect of CRH and α-helical CRH on IκB expression.

Fig. 3.

CRH Effect on IκBα Protein Expression Western blot analysis of whole cell protein from mouse thymocytes. A, Time-course of IκBα abundance in response to CRH (10⁻⁷m) treatment. Lane 1, Untreated cells (control). Lanes 2–5, CRH treatment for 15 min to 3 h. B, Effect of CRH receptor antagonist. Lane 1, CRH (10⁻⁷m) treatment for 30 min; lane 2, CRH-treated cells after 30 min pre-incubation with α-helical CRH (10⁻⁷m), lane 3, untreated cells. Results represent one of three individual experiments. C, Quantitative analysis of the effect of CRH and α-helical CRH on IκB expression from three individual experiments. *, P ≤ 0.01 compared with control.

Effect of CRH on NF-κB and IκBα Expression in a Mouse Thymus Cell Line

To obtain an easier-to-access-and-handle cell system than the primary culture of thymocytes, we evaluated the effects of CRH on NF-κB DNA binding activity and IκBα abundance in the mouse thymic lymphoma cell line, the WEHI 7.1 cells. CRH (10⁻⁷m) induced NF-κB DNA-binding activity in WEHI 7.1 cells similarly to its effect on the primary thymocytes, an effect blocked by α-helical CRH (Fig. 4A), whereas it reduced IκBα expression (Fig. 4B), as described above for the primary thymocytes. These findings suggest that WEHI 7.1 cells provide a good model for studying the effects of CRH on mouse thymocytes.

Fig. 4.

Effect of CRH on NF-κB Activation and IκBα Expression in WEH 7.1 Cells A, EMSA analysis of NF-κB activation. B, Quantitative analysis of the effect of CRH, LPS, and α-helical CRH coadded with CRH on the NF-κB DNA binding activity in WEH.1 cells. Results represent mean values ± sem from three different experiments. C, Western blot analysis of IκBα expression. Results represent one of three individual experiments. D, Supershift EMSA of the abundance of the NF-κB subunits, p50 and p65, in WEH 7.1 cells treated with CRH, or not (control). Results represent one of three individual experiments. n.s., Nonspecific band shown as an indicator of loading.

Abundance of p50 and p65 in the CRH-Induced NF-κB Complex

The abundance of p50 and p65, two major members of the Rel/NF-κB family, was evaluated by supershift assay in WEHI 7.1 cells (Fig. 4C). Addition of anti-p50 or anti-p65 antibody reduced the intensity of the NF-κB complex in either control (lanes 3 and 5) or CRH-treated cells (lanes 4 and 6), although the effect of the anti-p50 antibody in the CRH-treated cells was less than that of anti-p65 antibody. The above indicate that both p65 and p50 participate in the formation of the constitutive, as well as the CRH-induced NF-κB DNA-binding complex in mouse thymocytes. The observed differences in the effectiveness of the two antibodies might reflect relative differences in the constitution of the CRH-induced NF-κB complex. The possibility of additional members of the Rel/NF-κB family participating in the NF-κB DNA binding complex in these cells is not excluded by these data.

NF-κB Activation in CRH-Deficient Mice

We used wild-type (Crh+/+) and CRH-efficient (Crh−/−) mice (47) to evaluate the significance of endogenous CRH in the induction of NF-κB DNA binding activity in vivo. Plasma cytokine and corticosterone levels and thymic NF-κB DNA binding activity were evaluated 4 h after ip administration of LPS (100 μg). In agreement with our in vitro findings, LPS-induced NF-κB activation was much lower in the Crh−/− compared with the Crh+/+ mice (Fig. 5A2), whereas no differences were found in the constitutive (basal) NF-κB activation between the two genotypes (Fig. 5A2). Thus, CRH deficiency is related to decreased NF-κB activation after LPS, despite the concomitant increase in their plasma cytokine levels and their relative glucocorticoid insufficiency (Fig. 5, B and C; Ref. 58).

Fig. 5.

Thymus NF-κB Activation in Crh+/+ and Crh−/− Mice EMSA analysis of thymus NF-κB DNA-binding activity in basal conditions (A1) or 4 h following LPS administration (A2). B and C, Serum TNFα and corticosterone levels before (basal) and after LPS administration in Crh+/+ and Crh−/− mice. *, Significant difference (P < 0.05) from basal levels of the same genotype; #, significant difference (P < 0.05) between Crh+/+ and Crh−/− mice at the same time point. n.s., Nonspecific band shown as an indicator of loading.

DISCUSSION

Increasing evidence demonstrates the expression of CRH in a variety of inflammatory conditions, as well as its in vivo direct immunomodulatory effects, with reports suggesting its proinflammatory (3–5, 12–14), as well as its antiinflammatory actions (19–23). The biological significance of CRH expressed in immune organs such as the spleen and the thymus (6, 9, 10, 24) has not been elucidated yet. There are hypotheses that immune CRH stimulates the locally expressed proopiomelanocortin gene (30) and proinflammatory cytokines, and thus participates in the inflammatory process (3, 13) and the corresponding pain sensation (12, 31). Our findings demonstrating increased NF-κB DNA binding activity in mouse thymocytes after CRH treatment, provide a potential mechanism mediating the proinflammatory effects of peripheral CRH.

The stimulatory effect of CRH on the NF-κB DNA-binding activity in mouse thymocytes was exerted at concentrations as low as 10⁻¹¹m and reached a plateau at 10⁻⁷m. Furthermore, it was blocked by the nonspecific CRH antagonist α-helical CRH (Fig. 1C), whereas no effect was seen after administration of either α-helical alone or a specific CRH-R1 antagonist CP 154,526 (Zhao, J., and K. Karalis, unpublished observation). The latter suggests that endogenous CRH is unlikely to play a significant role on the constitutive expression of this effect of NF-κB. In addition, the above-described effect of CRH on NF-κB may be exerted by its binding on CRH-R2, which would be compatible with the effective dose range of CRH in this system. In addition, the likelihood of CRH-R2 mediating this effect of CRH raises the possibility that the other peptides of the CRH family that bind CRH-R2 and even with higher affinity that CRH, might exert similar effects. More studies are required to evaluate the relative concentrations of these peptides in immune tissues and their potential immunomodulatory properties. Inhibition of the NF-κB activation by CRH in pituitary cells that express only the CRH-R1, or in hippocampal cells transfected with the CRH-R1 has been reported recently (59). This finding, together with ours, suggests that the effect of CRH, stimulatory or inhibitory, on NF-κB binding activity might be tissue specific and thus, most likely, related to the CRH receptor subtype and the relative concentration of the different ligands of the CRH receptor(s) expressed in that tissue.

LPS and proinflammatory cytokines such as TNFα are potent immune system stimulators that induce NF-κB activity, which further propagates the inflammatory response (46, 48). In this study, we have shown that CRH induces NF-κB activity in vitro in a mode similar to LPS (Fig. 1). Addition of LPS on thymocytes pretreated for 30 min with LPS abolished any induction of the NF-κB DNA binding activity. This phenomenon has been previously described after cotreatment with LPS and proinflammatory cytokines that are naturally induced by LPS and has been attributed to desensitization (60, 61). Finally, induction of NF-κB activity by LPS in vivo was significantly compromised in states of CRH deficiency (Fig. 5A2). The above suggest that the LPS- and CRH-mediated induction of NF-κB activity might be operated by similar signaling pathways. In fact, the kinetic of the NF-κB activation induced by CRH is very similar to that of TNFα (62), the cytokine that represents the initial response to LPS (63).

Inhibition of NF-κB and increase of IκB after glucocorticoid treatment have been proposed to represent the major mechanism for the immunosuppressive effects of this steroid. Several studies have also shown that glucocorticoid inhibition of the cytokine-induced NF-κB activity is complex and most likely is both cell and stimulus specific (51, 52, 64–66). We found that dexamethasone pretreatment led to a significant suppression of both the CRH- and the LPS-induced NF-κB DNA binding activity in mouse thymocytes (Fig. 2).

The role of PKA in the regulation of NF-κB has been previously reported (56, 67), whereas blockade of the CRH-mediated induction of the NF-κB DNA binding activity by the PKA inhibitor H89 suggests the importance of this kinase in this process. The increase in the constitutive NF-κB DNA-binding activity after treatment with H7 is intriguing because it suggests that PKC activation might act as an inhibitor of the basal expression of NF-κB in thymocytes. Furthermore, the inability of CRH to further stimulate the NF-κB DNA binding activity in the presence of H7 suggests that its effects are mediated by the PKC signaling pathway, as has been shown for other immunomodulatory factors (68–70). The relative contribution of each of the two kinase-dependent signaling pathways in mediating the stimulatory effect of CRH on the NF-κB DNA binding activity remains to be assessed.

The physiological significance of the CRH-induced NF-κB DNA binding activity is the subject of on-going studies. Our preliminary data suggest that CRH induces the transcriptional activation of E-selectin, an inflammation-related NF-κB-responsive gene, in cells transfected with CRHR2 but not with CRHR1 and subsequently transiently transfected with a construct containing the promoter of E-selectin driving the luciferase reporter gene (van Vloerken, L., J. Gay, and K. P. Karalis, manuscript in preparation). Furthermore, our findings of decreased NF-κB DNA binding activity in the thymus of the Crh−/− mice compared with the Crh+/+ mice after LPS administration (Fig. 5), provide further support of the proinflammatory effects of CRH. It is interesting that Crh−/− mice had lower levels of NF-κB DNA-binding at the same time that their circulating levels of proinflammatory cytokines, such as TNFα and IL-1β, were two to four times higher and their corticosterone levels were significantly lower than in the Crh+/+ mice (Fig. 5 and Ref. 42). This finding suggests that the induction of the NF-κB DNA binding activity by CRH may be independent of the stimulatory effect of cytokines (46) and/or the inhibitory effect of glucocorticoid on NF-κB activation (52, 53).

In summary, we have demonstrated that in mouse thymocytes CRH induces the DNA-binding activity of NF-κB, a nuclear transcription factor critical for the regulation of cytokine and other inflammatory mediator genes. Our findings provide the first evidence for a molecular pathway that may mediate the proinflammatory effects of CRH. The implications of these findings might be important for inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease regulated by NF-κB and shown to be associated with increased CRH expression (2, 14, 15, 71).

MATERIALS AND METHODS

Cell Culture

Primary culture of mouse thymocytes.

Mouse thymus glands were dissected from 2- to 3-month-old female C57BL mice. Thymocytes were isolated by squeezing the thymuses between two glass slides followed by fractionation over discontinuous Histopaque 1070 gradient (Sigma, St. Louis, MO). Cells with densities between 1.060 and 1.070 were collected and washed once with Roswell Park Memorial Institute 1640 culture medium. Cells (2 × 10⁶/ml) were cultured in six-well culture plates in Roswell Park Memorial Institute 1640 medium (Life Technologies, Inc., Grand Island, NY), supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma) at 37 C in an atmosphere of 5% CO₂. On the next day, cells were treated as indicated in the figure legends.

WEHI 7.1 cell.

WEHI 7.1 cell, a mouse thymic lymphoma cell line, was purchased from ATCC (Manassas, VA). Cells were maintained in culture in high glucose DMEM (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 1% antibiotics (penicillin/streptomycin) (Sigma) at 37 C in an atmosphere of 5% CO₂. Culture medium was changed every 2–3 d.

Isolation of Nuclear Extracts

At the end of the experimental period, cells were harvested and cell pellets were lysed in 200 μl ice-cold hypotonic lysis buffer containing 10 mm HEPES-KOH (pH 7.9), 10 mm KCl, 1.5 mm MgCl₂, 0.2 mm EDTA, 0.5 mm dithiothreitol (DTT), 10 μg/ml each of aprotinin, leupeptin, and pepstatin, 1 mm NaF, 1 mm aVO₄, and 1% Nonidet P-40, for 10 min. After brief centrifugation at 3000 × g for 1 min, the cytosolic extracts were collected and the nuclear pellets were lysed in 40 μl of high salt extraction buffer containing 20 mm HEPES-KOH (pH 7.9), 0.42 m NaCl, 1.5 mm MgCl₂, 0.3 mm EDTA, 0.5 mm DTT, 20% glycerol, 0.1% Triton X-100, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin. After incubation on ice for 45 min with intermittent vortexing, the nuclear extracts were collected after centrifugation at 14,000 × g for 30 min at 4 C and their protein concentration was determined with the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL) using BSA as a standard. The nuclear extracts were stored at −80 C until further use.

DNA-Protein Binding Studies

Double-stranded oligonucleotides of the core sequence of the NF-κB binding element on mouse immunoglobulin κ light chain (sense: 5′ TCG ACA GAG GGG ACT TTC CGA GAC GC 3′; antisense: 5′ TCG AGC CTC TCG GAA AGT CCC CTC TG 3′) (32) were labeled with [³²P]deoxy-CTP (50 μCi at 3000 Ci/mmol from NEN Life Science Products, Boston, MA) using Klenow fragment of Escherichia coli DNA polymerase I (Roche Molecular Biochemicals, Indianapolis, IN). Equal amounts (6–10 μg) of nuclear extracts were incubated in 20 μl binding buffer containing 10 mm Tris-HCl, pH 7.5; 50 mm NaCl; 0.5 mm DTT; 0.5 mm EDTA; 1 mm MgCl_2^- MDNM⁻; 4% glycerol, 2.5 μg of poly(deoxyinosine/deoxycytidine) (Amersham Pharmacia Biotech), and 2 μl of the ³²P-labeled probe for 30 min at room temperature. In competition experiments, excess amount of unlabeled NF-κB oligonucleotide or unlabeled mutant NF-κB oligonucleotides with a G to C mutation were used as competitors. For the supershift studies, 2 μl of antibody (anti-p50, anti-p65, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the reaction mixture and incubated for another 20 min at room temperature before the addition of the ³²P-labeled probe. The DNA-protein binding complexes were analyzed by EMSA on a nondenaturing 6% polyacrylamide gel using a Tris/glycine/EDTA buffer. The dried gel was exposed to film at −70 C.

Whole Cell Protein Extraction and Western Blot Analysis

Cells collected at the end of treatment were lysed in buffer containing 150 mm NaCl, 25 mm Tris-Cl, 2 mm EDTA, 1 mm NaF, 1 mm NaVO₄, 1 mm phenylmethylsulfonylfluoride, 1% Nonidet P-40, and 10 μg/ml of aprotinin, leupeptin, pepstatin. Total cell protein was quantitated with the bicinchoninic acid protein assay kit (Pierce Chemical Co.). An equal amount (50 μg) of protein from each sample was loaded onto SDS-polyacrylamide gel for electrophoresis separation and then transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). After transfer, the gel was stained by Coomassie blue overnight to confirm equal loading and blotting. The membrane was blocked in 5% nonfat milk overnight at 4 C and incubated with anti-IκBα antibody (1: 200, Santa Cruz Biotechnology, Inc.) (SC1643) for 2 h at room temperature. After extensive washing, the blot was incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The specific protein band was then detected by the enhanced chemiluminescence method (enhanced chemiluminescence kit, Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

Animal Housing

Crh+/+ and Crh−/− (47) mice of 129xC57BL/6 genetic background were housed with ad libitum access to rodent chow on a 12-h light, 12-h dark cycle (lights on at 0700 h). Animal housing and care was done according to NIH guidelines and all experiments were approved by the Animal Care and Use Committee of Children’s Hospital in Boston. All experiments were performed in mice of 2–4 months age. Animals were housed individually at least 48 h before each experiment.

Animal Studies

LPS (100 μg) was administered by ip injection at 0800 h. Control animals received a similar injection of sterile normal saline. Blood samples were collected by retroorbital eye bleeding of conscious mice (4–5 mice/group) 4 h later. At the end of each experiment, mice were killed by decapitation and thymuses were harvested for preparation of nuclear extracts.

Plasma Hormone and Cytokine Assays

Blood samples were centrifuged at 3000 rpm (1925 × g) at 4 C for 10 min, and plasma was separated, aliquoted, and stored at −80 C until further use. Plasma ACTH (INCSTAR Corp., Stillwater, MN) and corticosterone (ICN Pharmaceuticals, Costa Mesa, CA) levels were measured using commercial RIA kits. Plasma TNFα concentrations were measured by commercial ELISA kit (R&D Systems Inc., Minneapolis, MN).

Reagents

Human/rat CRH was kindly provided by Dr. G. Chrousos (NICHD, Bethesda, MD). The NF-κB oligonucleotides were synthesized by Life Technologies, Inc. (Rockville, MD). LPS and α-helical CRH (amino acids 9–41) were purchased from Sigma. N-[2-(p-Bromocinnamylamino) ethyl]-5-isoquinolinesulphonamide (H89) and 1-(5-isoquinolinesulphonyl)-2-methyl-ipiperazine (H7) were purchased from ICN Pharmaceuticals. Dexamethasone was purchased from Elkins-Sinn (Cherry Hill, NJ). The antibodies were purchased from Santa Cruz Biotechnology, Inc.

Data Analysis

The specific bands from x-ray films from EMSAs were scanned by densitometer using the PhotoShop software and analyzed with the NIH Image software. Statistical significance of differences was calculated by ANOVA followed by Scheffé’s and Fisher’s least significant difference post hoc multiple comparison test.

Acknowledgments

The authors would like to thank Drs. Joseph Majzoub and George Pavlakis (National Cancer Institute, Frederick, MD) for insightful comments and technical assistance with EMSA.

This work was supported NIH Grant RO1-DK-47977 (to K.P.K.) and the Children’s Hospital Department of Medicine 1999 Research Scholar Award (to K.P.K.).

Abbreviations:

 
• DTT,

  Dithiothreitol;

 
• IκBα,

  inhibitor of NF-κB;

 
• LPS,

  lipopolysaccharide;

 
• NF-κB,

  nuclear factor-κB;

 
• PKA,

  protein kinase A;

 
• PKC,

  protein kinase C.