Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver

Abstract

We generated mice carrying a STAT3 allele amenable to Cre-mediated deletion and intercrossed them with Mx-Cre transgenic mice, in which the expression of Cre recombinase can be induced by type I interferon. Interferon-induced deletion of STAT3 occurred very efficiently (more than 90%) in the liver and slightly less efficiently (about 70%) in the bone marrow. Analysis of the induction of liver acute-phase genes in response to bacterial lipopolysaccharide unequivocally identifies STAT3 as a fundamental mediator of their induction. The different degrees of defectiveness displayed by the various genes allowed us to differentiate them into three separate groups according to their degree of dependence on STAT3. Induction was totally defective for group I genes, defective at 24 h but almost normal at earlier time points for group II genes, and only slightly defective for group III genes. This division was in good agreement with the known structures of the respective promoters. We also found that the overall induction of the transcription factors C/EBP beta and -delta was only minimally defective in the absence of STAT3. Finally, even though corticosterone levels and action were found to be normal in the conditional-mutant mice, production of both proinflammatory and antiinflammatory cytokines was increased and prolonged, probably as a result of STAT3 deletion in macrophages.

FIG. 1

Generation of STAT3^fl/+ ES cells and of MX⁺ or MX⁻ STAT3^fl/fl mice. (A) The structure of the replacement targeting vector along with the structures of the wild-type, replaced, floxed, and deleted alleles, the position of the probe used, and the predicted sizes of the restriction fragments are depicted. The replaced allele was detected by Southern blotting on EcoRV-digested genomic DNA using as a probe a cDNA fragment spanning exons 15 to 24. Cre-mediated deletion was expected to generate STAT3 floxed and deleted alleles and was diagnosed by Southern blotting using the intronic probe 2.1. (B, C, and D) Southern blot analysis of genomic DNA from targeted ES cells either before (B and C) or after (D) Cre-mediated deletion. The probes used and the relevant alleles detected are indicated (wt, wild type; repl, replaced; fl, floxed; del, deleted). (E and F) Western blot of liver extracts from poly(I · C)-treated mice. MX⁺ or MX⁻ STAT3^fl/fl mice were injected once i.p. with poly(I · C) followed 4 days later by either LPS or apyrogenic saline solution. The livers from the indicated mice were collected after 4.5 and 9 h (F) or after 24 h (E), and total extracts were subjected to Western blot analysis with anti-STAT3 (E) or anti-phospho-(Tyr 705) STAT3 (P-STAT3) (F) antibodies. The blots were stripped and reprobed with anti-STAT1 (E) or anti-STAT3 (F) antibodies. C, STAT3-enriched control extract.

FIG. 2

LPS induction of AP mRNAs as measured by slot blot analysis on STAT3 conditional-mutant (open bars) and control (solid bars) mice. The mice were injected with either LPS or saline solution 4 days after poly(I · C) treatment, and their livers were collected after 0, 1.5, 6, 12, and 24 h, as indicated. Total RNA was extracted, denatured, transferred to nylon membranes by slot blotting, and hybridized with the indicated cDNA probes. The results, shown as mean values + standard errors of the mean of at least five mice per group, were plotted after normalization with GAPDH as an internal control. All values obtained after saline injection were uniform and were therefore pooled and shown collectively as time zero, representing the steady-state value.

FIG. 3

IL-6 induction of AP mRNAs as measured by slot blot analysis on STAT3 conditional-mutant (open bars) and control (solid bars) mice. The mice (three per group) were injected with either recombinant IL-6 or saline solution 4 days after poly(I · C) treatment, and their livers were collected after 0, 1.5, 6, and 24 h, as indicated. The methods and symbols are described in the legend to Fig. 2.

FIG. 4

Induction of C/EBPβ and -δ as measured by Northern blot, Western blot, and EMSA analyses. (A and B) STAT3 conditional-mutant (open bars) and control (solid bars) mice were treated for the indicated times as described in the legend to Fig. 2 with either LPS (A) or recombinant IL-6 (B) and analyzed by Northern blotting. The data are shown as mean values + standard errors of five (A) or three (B) mice per group. The values were normalized and plotted as described in the legend to Fig. 2. The symbols indicate statistically significant differences between the two groups of mice at each time point: #, P < 0.03; ∗, P < 0.01; ‡, P < 0.0001. (C) Western blot analysis of C/EBPβ and C/EBPδ. Mice were treated with LPS as described above for 4.5 or 9 h, and nuclear extracts were analyzed by Western blotting with specific antisera. The different polypeptides detected are indicated. −, negative; +, positive. (D) EMSA analysis of C/EBP binding activities. Nuclear extracts from mice either untreated or treated with LPS for 4.5 h were used in an EMSA with a double-stranded oligonucleotide carrying a C/EBP binding site. Where indicated, antisera against different C/EBP proteins were preincubated with the extracts.

FIG. 5

(A and B) Serum cytokine and CS levels measured in LPS-treated STAT3 conditional-mutant (open bars) and control (solid bars) mice. The mice were treated as described in the legend to Fig. 2. Blood was collected at the indicated time points, and the cytokine content of the serum was measured by ELISA (A) or the CS content was measured by radioimmunoassay (B). The data are shown as mean values + standard errors of the mean of at least five (A) or three (B) mice per group. The symbols indicate statistically significant differences between the two groups of mice at each time point: #, P < 0.01; ∗, P < 0.001. (C) Cytokine levels upon pretreatment with dexamethasone. Mice were treated with either dexamethasone or apyrogen saline solution 30 min prior to LPS injection, and blood was collected 1 h later and subjected to ELISA analysis. +, present; −, absent.

FIG. 6

EMSAs and anti-MAPK detection with liver extracts from STAT3 conditional-mutant and control mice. The mice were treated as described in the legend to Fig. 2. (A) Livers were collected 4.5 and 9 h after LPS injection, and nuclear extracts were prepared immediately. Total nuclear extracts were incubated with ³²P-labeled double-stranded oligonucletides carrying binding sites for NF-κB (from the human immunodeficiency virus 3′ long terminal repeat) or STAT3-STAT1 (corresponding to the sis-inducible element on the c-fos promoter). (B) To detect MAPK phosphorylation, total extracts from mice treated with LPS for the indicated lengths of time were subjected to Western blot analysis with anti-phospho-(p^44/42) MAPK antibodies (P-MAPK). The blots were stripped and reprobed with anti-MAPK antibodies (MAPK). +, positive; −, negative.

FIG. 7

(A) Structure of the analyzed AP promoters from the corresponding mouse (m), rat (r), or human (h) genes. Open ovals, C/EBP–type I IL-6RE sites; solid rectangles, STAT–type II IL-6RE sites; shaded rectangles, proposed STAT sites which have not been functionally characterized; dotted oval on the rat FBα promoter, potential C/EBP site that was found not to be functional. The position and sequence of each STAT element is shown, and previous nomenclature is also given when appropriate. The coordinates of each promoter are indicated, and elements identified on the promoters but not involved in cytokine responsiveness are named and indicated by arrows. Both the distal (DRE) and the Proximal (PRE) regulatory regions of the rat AGP gene are shown. The glucocorticoid responsive element (GRE) contained within the PRE is required for optimal induction. References are given in the text. (B) Sequence comparison of the C/EBP sites on the human, mouse, and rat Hpx promoters. Bases mutated in the mouse and rat promoters compared to the human sequence are in boldface and underlined.