Viable Model Inflammatory Responses in the Motheaten

Activated Ets2 Is Required for Persistent Inflammatory Responses in the Motheaten Viable Model

This information is current as Guo Wei, Jianping Guo, Andrea I. Doseff, Donna F. of September 29, 2021. Kusewitt, Albert K. Man, Robert G. Oshima and Michael C. Ostrowski J Immunol 2004; 173:1374-1379; ; doi: 10.4049/jimmunol.173.2.1374

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activated Ets2 Is Required for Persistent Inﬂammatory Responses in the Motheaten Viable Model1

Guo Wei,* Jianping Guo,* Andrea I. Doseff,*‡ Donna F. Kusewitt,† Albert K. Man,¤ Robert G. Oshima,¤ and Michael C. Ostrowski2*‡

The Ets2 transcription factor is constitutively phosphorylated on residue Thr72 in macrophages derived from mice homozygous for the motheaten viable (me-v) allele of the hemopoietic cell phosphatase (Hcph) gene. To genetically test the importance of signaling through residue Thr72 of Ets2 during inflammation, the Ets2A72 mutant allele, which cannot be phosphorylated on Thr72, was combined with the Hcphme-v allele in mice. Ets2A72/A72 moderated the inflammation-related pathology of Hcphme-v/me-v mice, as demonstrated by the increased life span and the decreased macrophage infiltration in skin and lungs of these mice. Macrophage apoptosis induced by cytokine withdrawal was also increased in the double-mutant mice. Importantly, the Ets2A72/A72 allele

␣ ␣ Downloaded from resulted in decreased expression of inflammatory response genes, including TNF- , CCL3, matrix metalloprotease 9, integrin M, and Bcl-X in alveolar macrophage. Ets2 phosphorylation was required for persistent activation of TNF-␣ following LPS stimulation of bone marrow-derived macrophages. The phosphorylation of Ets2 functions in the severe inflammatory phenotype of the me-v model by mediating both macrophage survival and inflammatory gene expression. The Journal of Immunology, 2004, 173: 1374Ð1379.

he ETS family, consisting of 28 members in the human mutation is introduced into the mouse germline, it behaves as a http://www.jimmunol.org/ genome, encodes for sequence-specific DNA-binding hypomorphic allele that is haploinsufficient for embryonic devel- T proteins that are transcriptional activators and repressors opment, but homozygous viable (10). (1). The family is defined by a highly conserved DNA binding To define the biological role of Ets2 phosphorylation, we have domain referred to as the ETS domain. However, the DNA-bind- previously studied Ets2 activation in macrophages, particularly in ing properties of these factors are similar and cannot entirely ac- the context of the motheaten viable (hemopoietic cell phosphatase count for the specificity required for the precise activation of target (Hcphme-v/me-v)) mouse mutation (11). We demonstrated that Ets2 genes that occurs during the diverse biological processes mediated was constitutively phosphorylated on residue Thr72 by a PI3K/jun by individual family members. Modification of discrete family N-terminal kinase pathway in the Hcphme-v/me-v model of acute members by signal transduction pathways provides an additional inflammation, in contrast to wild-type cells, in which Ets2 phos- by guest on September 29, 2021 mechanism to determine specificity (1). phorylation is dependent on cytokine action, for example CSF-1. For example, phosphorylation of the ETS family members Ets1 Furthermore, constitutive Ets2 activation correlated with expres- and Ets2 by ras-dependent pathways leads to persistent expression sion of target genes such as Bcl-X and uPA3 and with the increased of target genes, including extracellular proteases such as urokinase survival of Hcphme-v/me-v macrophages. To directly test the role of plasminogen activator (uPA) and stromelysin/matrix metallopro- Ets2 phosphorylation in the pathology of Hcphme-v/me-v mice, we tease-3 (MMP-3) (2, 3). Ets1 and Ets2 are phosphorylated at a combined the Ets2A72/A72 allele with Hcphme-v/me-v genes in mice. conserved residue (Thr38 and Thr72, respectively) by the well-char- This genetic analysis demonstrates the importance of MAPK ac- acterized ras-effector pathway, the Raf/MAPK pathway (4Ð6). tivation of Ets2 in the chronic inflammatory responses character- The key phosphorylation event occurs within a region of Ets1 and istic of Hcphme-v/me-v mice such as TNF-␣ induction, and confirms Ets2 that is conserved through evolution with the Drosophila P2 a key role for Ets2 in mediating CSF-1 survival signaling in protein, and has been termed the pointed domain (7, 8). This region macrophages. of homology plays a role in protein-protein interactions, both ho- mophilic and with other proteins (1, 9). Changing the codon for Materials and Methods 72 A72 Thr in the Pointed domain of Ets2 to alanine (Ets2 ) results in Mice a gene product with lowered trans activation potential especially T72A tmA72Osh A72 evident in the presence of ras signaling (2Ð6). When the Ets2A72 The Ets2 mutation (Ets2 referred to in this work as Ets2 ) was generated by knockin gene targeting, as described (10), and a congenic FVB/N line carrying this mutation was created by nine generations of A72/A72 † backcross. Ets2 mice are viable and fertile, but limit the growth of Departments of *Molecular Genetics and Veterinary Biosciences, and Comprehen- me-v sive Cancer Center, and ‡Dorothy Davis Heart and Lung Institute, Ohio State Uni- mammary tumors (10). The Hcph mutation was originally obtained in versity, Columbus, OH 43210; and ¤Burnham Institute, La Jolla, CA 92037 the C57BL/6J background from The Jackson Laboratory (Bar Harbor, ME) and a congenic FVB/N line established after nine generations of back- Received for publication February 19, 2004. Accepted for publication May 11, 2004. crosses. The phenotype of Hcphme-v mice in the FVB/N background was The costs of publication of this article were defrayed in part by the payment of page identical with that reported for the mutation in the C57BL/6J background charges. This article must therefore be hereby marked advertisement in accordance (12). All mice were genotyped by PCR. Primers and conditions used are with 18 U.S.C. Section 1734 solely to indicate this fact. available upon request. Use and care of mice in this study were approved 1 This work was supported by National Cancer Institute Grants R01-CA53271 (to M.C.O.), R01-CA74547 (to R.G.O.), and R01-CA98778 (to R.G.O.). 2 Address correspondence and reprint requests to Dr. Michael C. Ostrowski, Depart- 3 Abbreviations used in this paper: uPA, urokinase plasminogen activator; BMM, ment of Molecular Genetics, Ohio State University, Riffe Building, Room 834, 406 bone marrow-derived macrophage; Hcph, hemopoietic cell phosphatase; me-v, moth- West 12th Avenue, Columbus, OH 43210. E-mail address: [email protected] eaten viable; MMP, matrix metalloprotease.

ϩ ϩ by the Ohio State University Institutional Animal Care and Use compared with Hcphme-v/me-v mice with wild-type Ets2 (Ets2 / ; Committee. Hcphme-v/me-v), with 75% of the former group surviving (n ϭ 37), Cell culture compared with 12% of the latter (n ϭ 35; Fig. 1A). The body weight of the Ets2A72/A72; Hcphme-v/me-v mice was approximately Bone marrow-derived macrophages were obtained, as described (11). The ϩ/ϩ me-v/me-v collection of thioglycolate-elicited peritoneal macrophages was described twice that of Ets2 ; Hcph mice, and indistinguishable previously (13). The lavage fluid was centrifuged, and the cells were plated from wild-type mice of the same age and genetic background (Fig. in six-well dishes (nontissue culture treated). Thirty minutes later, the un- 1B). The Ets2A72/A72; Hcphme-v/me-v mice, both males and females, attached cells were removed and total RNA was extracted from attached were also fertile, in contrast to Ets2ϩ/ϩ; Hcphme-v/me-v mice, which macrophages. The collection of murine alveolar macrophage was con- were never fertile, as reported during the original characterization ducted, as described (14). The lung alveolar cells were lavaged, centrifuged, and resuspended in 1 ml of RPMI 1640 medium with 0.1% heat- of these mice (12). inactivated calf serum, and transferred to a well of a 24-well dish One major immunopathological abnormality in Hcphme-v/me-v (nontissue culture treated). Six hours later, nonadherent cells were removed mice is the accumulation of macrophages and granulocytes in the and RNA was prepared from the adherent macrophages. dermis with inflammatory cells penetrating as far as the panniculus Cell survival assays pigment, giving the mice their characteristic motheaten appear- ance. Additionally, inflammation in the limbs and joints is asso- The viability assay was conducted using the LIVE/DEAD Viability/Cyto- toxicity kit (Molecular Probes, Eugene, OR), using procedures described ciated with an arthritis-like condition in the joints (19). This type by the manufacturer. DNA fragmentation assay was performed, as de- of inflammation is especially severe in both the feet and digits of scribed (15). The fragmented DNA from apoptotic cells was mixed with

SyBr Green (Molecular Probes), and analyzed in 1.2% agarose gels. The Downloaded from gel was visualized under UV light in a gel imager (Bio-Rad, Hercules, CA). The caspase 3 assay was performed exactly as described (16). Quantitative real-time PCR Total RNA was extracted from cells and tissues by TRIzol (Invitrogen Life Technologies, Carlsbad, CA). DNA was degraded by DNase I (Roche, Basel, Switzerland) digestion, and RNA was purified by RNeasy mini kit http://www.jimmunol.org/ (Qiagen, Valencia, CA). For lung alveolar macrophage, RNA was extracted with RNeasy mini kit column (Qiagen), and DNA was removed by on-column DNase I (Qiagen) digestion. Total RNA was reverse transcribed by Superscript III reverse transcriptase (Invitrogen Life Technologies) with random hexamer primers. Primers used for real-time PCR were picked by Primer 3 software (17), and sequences used are available upon request. The real-time PCR was conducted using SyBr Green super mix (Bio-Rad) in an Icycler iQ Real-Time Detection system (Bio-Rad). The PCR threshold was determined by Icycler PCR baseline subtracted curve fit method. The threshold for the gene being studied was adjusted by that of a reference gene (either ribosomal protein L4 gene or lysozyme, as indicated in by guest on September 29, 2021 figure legends). The raw mRNA copy number was calculated as: 1/2(adjusted threshold). The relative copy number was obtained by dividing each raw mRNA copy number with the reference sample raw mRNA copy number. Results Increased viability and reduced pathology in Hcphme-v/me-v mice homozygous for the Ets2A72 allele The mouse mutant me-v (Hcphme-v/me-v) results from a point mutation that affects splicing of transcripts encoded by the hemopoietic cell phosphatase gene (Hcph, also termed src homology 2 tyrosine phosphatase-1), and leads to expression of a protein with 5Ð10% of wild-type tyrosine phosphatase activity (18). These mutant mice accumulate massive numbers of macrophages and neutrophils in the peripheral tissues, including skin, spleen, and lung, and subsequently succumb to an interstitial pneumonia (12). Thus, Hcph apparently plays a central role in cell signaling events that regulate macrophage-dependent inflammatory responses. In previous work, we demonstrated that the transcription factor Ets2 was constitutively phosphorylated and activated in macrophages isolated from Hcphme-v/me-v mice, and that Ets2 activation correlated FIGURE 1. Increased viability and reduced inflammation in Hcphme-v/me-v with expression of target genes such as Bcl-X and increased cell mice homozygous for the Ets2A72 allele. A, Cumulative survival of survival following growth factor withdrawal (11). To directly test Hcphme-v/me-v mice with wild-type or mutant Ets2A72. Data were based on n ϭ A72/A72 me-v/me-v ϩ/ϩ me-v/me-v the role of Ets2 activation in the pathology of Hcphme-v/me-v mice, 37 Ets2 ; Hcph mice and n ϭ 35 Ets2 ; Hcph mice. B, me-v/me-v ϭ we used a recently created Ets2A72 knockin allele that produces a Body weight of Hcph mice. The data reflect the average weight of n 6 mice from each genotype at 3 mo of age. Error bars indicate SD. Ⅺ, protein product that cannot be phosphorylated by ras-dependent ϩ ϩ wild-type mice; 1, indicates Ets2 / ; Hcphme-v/me-v mice; o, indicates signaling and is consequently a weak trans activator in studies Ets2A72/A72; Hcphme-v/me-v mice, respectively. C and D, Image of front performed in cell culture (2Ð6), and acts as a hypomorphic allele paws from representative Ets2ϩ/ϩ; Hcphme-v/me-v mice (C)orEts2A72/A72; in vivo (10). Hcphme-v/me-v mice (D). E–H, H&E-stained sections from front paws of A72 me-v Mice homozygous for both Ets2 allele and Hcph allele Ets2ϩ/ϩ; Hcphme-v/me-v mice (E and G) or the Ets2A72/A72; Hcphme-v/me-v mice (Ets2A72/A72; Hcphme-v/me-v) showed increased survival at 100 days (F and H). 1376 GENETIC INTERACTIONS BETWEEN Ets2 AND Hcph

Ets2ϩ/ϩ; Hcphme-v/me-v (Fig. 1C). The Ets2A72/A72; Hcphme-v/me-v To quantify the accumulation of macrophages in lung, RNA was mice were observed to lack severe skin and joint inflammation extracted from whole lungs from 90-day-old animals. Real-time (Fig. 1D). quantitative PCR was used to measure the expression of two mac- Histological analysis of sections made from the feet and digits rophage marker genes, c-fms and lysozyme (Fig. 2G). This analysis confirmed that inflammation was attenuated in the Ets2A72/A72; revealed that Ets2ϩ/ϩ; Hcphme-v/me-v mice had 3-fold higher levels Hcphme-v/me-v mice (Fig. 1, E–H). In Ets2ϩ/ϩ; Hcphme-v/me-v mice, of expression of c-fms and lysozyme compared with either wild- the epidermis was markedly thickened, and large areas of the der- type or Ets2A72/A72; Hcphme-v/me-v mice, results consistent with the mis were heavily infiltrated by mixed inflammatory cells (see re- histology results. gion indicated by arrowhead; Fig. 1E). There were numerous in- Increased apoptosis following CSF-1 withdrawal in macrophage traepidermal pustules in the epidermis, some of which had me-v/me-v A72 ruptured to the skin surface. In many areas, suppuration extended derived from Hcph mice homozygous for the Ets2 from the epidermis deep into the dermis. In addition, there was allele evidence of extensive remodeling of the cortices of the long bones Our previous work demonstrated a correlation between Ets2 phos- in the digits (arrow in Fig. 1G). This remodeling was due both to phorylation at Thr72 and increased survival of bone marrow-de- bone resorption and new bone formation and resulted in skeletal rived macrophages (BMMs) from Hcphme-v/me-v mice upon CSF-1 deformity. In marked contrast, the epidermis of the Ets2A72/A72; withdrawal (11). To directly test the role of Ets2 phosphorylation Hcphme-v/me-v mice was only two to three cell layers thick, iden- in the increased survival of BMMs from Ets2ϩ/ϩ; Hcphme-v/me-v, tical with the thickness for normal mice, and the dermis did not we used three different assays to study apoptosis of BMMs fol- contain large numbers of inflammatory cells (area indicated by lowing CSF-1 withdrawal: calcein acetoxymethyl/ethidium bro- Downloaded from arrow in Fig. 1F). Cortical surfaces of long bones were smooth, mide staining (Fig. 3A), DNA fragmentation assays (Fig. 3B), and and cortices were of uniform thickness (arrow in Fig. 1H). The caspase 3 assays (Fig. 3C). All three assays confirmed our previous skin, bones, and joints of the Ets2A72/A72; Hcphme-v/me-v mice were results showing that the BMMs from Ets2ϩ/ϩ; Hcphme-v/me-v mice indistinguishable from those of normal mice. are refractory to apoptosis induced by CSF-1 withdrawal when The accumulation of macrophages and neutrophils in lungs re- compared with wild type. The calcein AM/ethidium bromide assay ϩ/ϩ me-v/me-v sults in a fatal pneumonitis in Ets2 ; Hcph mice (12). At and DNA fragmentation assay also demonstrated that BMMs pre- http://www.jimmunol.org/ the histological level, this correlated with a massive accumulation pared from Ets2A72/A72; Hcphme-v/me-v mice had a significant 2- to of macrophages and neutrophils in the alveolar spaces in lungs of 3-fold increase in apoptosis (Fig. 3, A and B). These cells had a Ets2ϩ/ϩ; Hcphme-v/me-v mice (Fig. 2, C and D; arrowheads indicate 30-fold increase in caspase 3 activation following CSF-1 with- mononuclear cells within the alveolar space) compared with wild- drawal compared with cells from Ets2ϩ/ϩ; Hcphme-v/me-v mice type littermates (Fig. 2, A and B). However, there was little evi- (Fig. 3C). However, the BMM from Ets2A72/A72; Hcphme-v/me-v dence of macrophage accumulation in the alveolar spaces of lungs mice still had 2- to 3-fold less apoptosis and 3-fold lower caspase from Ets2A72/A72; Hcphme-v/me-v mice (Fig. 2, E and F). The lung 3 activation when compared with wild-type mice. sections from the double mutant looked similar to those of wild- The proliferation of BMMs from Ets2A72/A72; Hcphme-v/me-v mice type mice. The results shown are representative of results obtained was also compared with that of cells from Ets2ϩ/ϩ; Hcphme-v/me-v by guest on September 29, 2021 from nine Ets2A72/A72; Hcphme-v/me-v mice examined at age 90 days mice, but no significant difference was observed between the two or older, while all Ets2ϩ/ϩ; Hcphme-v/me-v mice (Ͼ10 examined) genotypes (Fig. 3D), indicating that Ets2 was not necessary for cell had similar lung pathology beginning at 50Ð60 days of age. proliferation.

FIGURE 2. Reduced lung inflammation of Hcphme-v/me-v mice homozygous for the Ets2A72 allele. Representative lung sections from wild-type (A and B), Ets2ϩ/ϩ, Hcphme-v/me-v (C and D), and Ets2A72/A72; Hcphme-v/me-v (E and F) mice. Arrowheads show examples of mononuclear cells within the alveolar space (C), or the absence of these cells in Ets2A72/A72 mice (E). B, D, and F, Originally ϫ100 magnification, and A, C, and E, originally ϫ400 magnification, respectively. G, Relative mRNA levels of macrophage markers of lungs from wild-type control (Wt); Ets2ϩ/ϩ; Hcphme-v/me-v (M), and Ets2A72/A72; Hcphme-v/me-v (AM) mice. Total RNA was isolated from freshly dissected lungs, reverse transcribed, and then subjected to real-time PCR analysis. The expression levels of each gene were standardized to the level of ribosomal protein L4 mRNA. The data represent the average obtained from two individual mice of each genotype, with each experiment performed in duplicate. Error bars indicate SD of measurements. The Journal of Immunology 1377

Inflammatory response gene expression is decreased in lungs and macrophage isolated from Hcphme-v mice homozygous for the Ets2A72 allele To further define the molecular basis underlying the attenuation of the inflammatory phenotype of Ets2A72/A72; Hcphme-v/me-v mice, quantitative real-time PCR was used to study gene expression in lungs obtained from mice of the three different genotypes used for these studies (Fig. 4). For this analysis, a set of well-defined target genes of Ets2, including extracellular proteases MMP1, MMP3, MMP9, uPA, and the antiapoptotic gene Bcl-X (2Ð4, 11, 20), was studied (Fig. 4A), as well as genes involved in macrophage-inflammatory responses, such as genes encoding cytokines (Fig. 4B), chemokines (Fig. 4C), and adhesion molecules (Fig. 4D). In this analysis, a number of genes were identified that were more highly expressed in lungs obtained from Ets2ϩ/ϩ; Hcphme-v/me-v vs wild-type mice. Most of these genes were also down-regulated in Ets2A72/A72; Hcphme-v/me-v mice. In particular, defined Ets2 targets such as MMP1, MMP9, uPA, IL-12, and Downloaded from TNF-␣ (21) were all genes that were down-regulated in the mice homozygous for the Ets2A72 allele. Additionally, genes known to have ets sites in their promoters, but with no evidence that they are direct targets for Ets2 such as cytokine gene IL-1␣, chemokine genes CCL3 and CCL4 (MIP-1␣ and MIP-1␤), and integrin genes ␣ ␤ ϩ/ϩ M and 2 (20), were all expressed at higher levels in Ets2 ; FIGURE 3. Phosphorylation of Ets2 is critical for macrophage survival. Hcphme-v/me-v than in wild-type or Ets2A72/A72; Hcphme-v/me-v mice. http://www.jimmunol.org/ Presented are the effects of CSF-1 withdrawal from BMMs of the three Expression of other genes, such as MMP3 (stromelysin), integrin genotypes studied: wild-type control (Wt); Ets2ϩ/ϩ; Hcphme-v/me-v (M), and ␤ , CCL5 (RANTES), and in particular Bcl-X, was not different Ets2A72/A72; Hcphme-v/me-v (AM), respectively. See Materials and Methods 3 for details of these assays. A, Cell viability assays using calcein AM/ between the three genotypes studied (Fig. 4A). ethidium bromide (live/dead) staining. Shown are the percentage of cells To determine whether expression differences in lung tissue re- surviving as a function of time following CSF-1 withdrawal. B, DNA frag- flected differences in gene expression in macrophages specifically, mentation assays. DNA was extracted 24 h after CSF-1 withdrawal and alveolar macrophages were prepared and expression of key genes analyzed using agarose gel electrophoresis of extracted DNA. MK is the was identified in the previous analysis determined (Fig. 5A). With

lane that contained DNA size markers. C, Caspase 3 assays. Assays were a few exceptions, the same genes were found to be differentially by guest on September 29, 2021 performed on cell extracts 24 h after CSF-1 withdrawal. D, Cell prolifer- expressed in alveolar macrophages. In particular, cytokine genes ation assays, determined by counting cells for the indicated times following TNF-␣ and IL-1␣, chemokine genes CCL2 and CCL3, integrin initial CSF-1 treatment. The data presented for the viability assays (A), genes ␣ , and extracellular protease genes MMP9 and uPA were caspase 3 assays (C), and proliferation assays (D) represent the average M all expressed at signiﬁcantly higher levels in cells isolated from results for cells obtained from three individual mice of each genotype. ϩ/ϩ me-v/me-v A72/A72 Error bars in each of the panels indicate the SD of the measurements. A Ets2 ; Hcph than from either wild-type or Ets2 ; me-v/me-v result representative of three independent DNA fragmentation experiments Hcph mice (Fig. 5A). In addition, Bcl-X was expressed at is shown in B. 4-fold higher levels in alveolar macrophage obtained from Ets2ϩ/ϩ; Hcphme-v/me-v than from Ets2A72/A72; Hcphme-v/me-v.

FIGURE 4. Reduced expression of in- ﬂammatory genes in lungs of Hcphme-v/me-v mice homozygous for the Ets2A72 allele. To- tal RNA was isolated from freshly dissected lungs from wild-type control (Wt), Ets2ϩ/ϩ; Hcphme-v/me-v (M), and Ets2A72/A72; Hcphme-v/me-v (AM) mice, respectively, then reverse transcribed, and subjected to real- time PCR analysis. The genes and genotypes studied are as indicated in the ﬁgure panels. The expression levels of genes encoding extracellular proteases and Bcl-X (A), cytokines (B), chemokines (C), and in- tegrins (D) were standardized to the level of lysozyme mRNA. The data presented are the average of two experiments performed in duplicate with lungs from two independent mice from each genotype. Error bars indicate the SD. 1378 GENETIC INTERACTIONS BETWEEN Ets2 AND Hcph

absolute levels of gene expression between the macrophage populations. BMMs do not express high levels of inﬂammatory response genes, but LPS treatment can stimulate such responses, for example, the expression of TNF-␣. We compared LPS induction of TNF-␣ in BMMs derived from wild-type, Ets2ϩ/ϩ; Hcphme-v/me-v, and Ets2A72/A72; Hcphme-v/me-v mice (Fig. 5C). This analysis demonstrated that early induction of TNF-␣ expression was indistinguishable in cells of the three different genotypes: after 30 min of LPS treatment, TNF-␣ mRNA levels were uniformly induced ϳ150-fold compared with untreated cells. However, after 18 h of LPS treatment, TNF-␣ mRNA expression remained 3- to 4-fold higher in Ets2ϩ/ϩ; Hcphme-v/me-v than in either wild-type or Ets2A72/A72; Hcphme-v/me-v cells, in which the level of expression had returned to near baseline activity. Therefore, persistent activation of TNF-␣ in Hcphme-v/me-v cells depended on phosphorylation of Ets2.

Discussion Downloaded from The results presented in this work provide genetic evidence dem- onstrating that Ets2 is necessary for the pathology of Hcphme-/me-v mutant mice, extending previous correlations between Ets2 and Hcph action surmised by biochemical and cell biological ap- proaches (11). As depicted in Fig. 6, Hcph acts as a negative reg-

ulator upstream of MAPK pathways that can lead to phosphory- http://www.jimmunol.org/ lation and activation of Ets2 in macrophages. The Hcphme-v allele encodes a product no longer capable of down-regulating signals that lead to MAPK activation, including activation of jun N-terminal kinase in macrophages (11), thus leading to constitutive activation of Ets2. Activation of Ets2 leads to increased expression of genes encoding cytokines, chemokines, extracellular proteases, and adhesion molecules, all involved in various aspects of inflam- FIGURE 5. Reduced expression of inflammatory genes in macrophage mation. Ets2 also directly regulates Bcl-X (11, 22), and thus de- isolated from Hcphme-v/me-v mice homozygous for the Ets2A72 allele. Real- creases apoptosis of activated macrophages. by guest on September 29, 2021 time PCR was used to determine the gene expression levels in macrophage derived from mice of the three genotypes: wild-type control (Wt), Ets2ϩ/ϩ; Hcphme-v/me-v (M), and Ets2A72/A72; Hcphme-v/me-v (AM). Both genes and genotypes studied are as indicated in the figure panels. A, RNA prepared from isolated lung alveolar macrophage. B, RNA prepared from isolated peritoneal macrophage. C, Expression of TNF-␣ in BMMs after LPS stimulation. Gene expression was standardized to the level of lysozyme mRNA for all of these experiments. The data presented for all experiments were the average result obtained with RNA from macrophages from two independent animals for each genotype, and each measurement was performed in duplicate. Error bars indicate the SD of the measurements.

These data indicate that Bcl-X was a target of Ets2 in macrophages, as previously demonstrated (11, 22), but not in all of the many other cell types present in lung tissue. To test whether the changes in gene expression observed were limited to lung macrophages or reflected a general change in tissue macrophage gene expression, expression of these genes was compared in peritoneal macrophages prepared from mice with the three different genotypes (Fig. 5B). The differential expression of genes between cells derived from Ets2ϩ/ϩ; Hcphme-v/me-v and Ets2A72/A72; Hcphme-v/me-v mice, including for Bcl-X, was similar in the two macrophage populations (Fig. 5B). However, there were differences in the relative levels of expression of several of these FIGURE 6. Model for Ets2 action in macrophage inflammation. Muta- genes in peritoneal compared with alveolar macrophages. For ex- tion of the Hcph gene results in constitutive activation of MAPK signaling ample, MMP9 was expressed at higher relative levels in alveolar pathways and constitutive activation of Ets2. This leads to activation of cells, while CCL2 was expressed at higher relative levels in peri- gene targets, both direct Ets2 targets and indirect targets, that contribute to toneal cells. In addition, IL-12 and MMP1 expression were more various aspects of cell inflammation. When the hypomorphic Ets2A72 allele dramatically different between the genotypes in peritoneal cells is present, expression of these target genes is reduced, and pathology due than alveolar (Fig. 5B). This might reflect inherent differences in to inflammation is reduced. The Journal of Immunology 1379

As predicted by this model, a mutation in the negative regulator 3. Wasylyk, C., A. P. Bradford, A. Gutierrez-Hartmann, and B. Wasylyk. 1997. Hcph is masked by the hypomorphic Ets2A72 allele, resulting in Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2. Oncogene 14:899. decreased expression of these inflammatory response genes, in- 4. Fowles, L. F., M. L. Martin, L. Nelsen, K. J. Stacey, D. Redd, Y. M. Clark, creased apoptosis, and a subsequent attenuation of acute inflam- Y. Nagamine, M. McMahon, D. A. Hume, and M. C. Ostrowski. 1998. Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphor- mation. Several of the affected genes are well-defined target genes ylation in response to colony-stimulating factor 1/c-fms signaling. Mol. Cell. Biol. of Ets2, including MMP1, MMP9, uPA, TNF-␣, and IL-12 (2Ð4, 18:5148. 11, 20, 21). Other affected genes, including CCL3, CCL4, and 5. McCarthy, S., D. Chen, B. Yang, J. Garcia Ramirez, H. Cherwinski, X. Chen, ␣ ␤ M. Klagsbrun, C. Hauser, M. Ostrowski, and M. McMahon. 1997. Rapid phos- integrin genes M and 2, contain ets sites in their cis-regulatory phorylation of Ets-2 accompanies mitogen-activated protein kinase activation and regions, although there is no direct evidence that Ets2 directly the induction of heparin-binding epidermal growth factor gene expression by regulates them (20). Overall, these data suggest that Ets2, either oncogenic Raf-1. Mol. Cell. Biol. 17:2401. 6. Rabault, B., M. F. Roussel, C. T. Quang, and J. Ghysdael. 1996. Phosphorylation directly or indirectly, regulates a set of genes that are necessary for of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogen- the severe inflammatory phenotype in the me-v model. esis. Oncogene 13:877. TNF-␣ plays a critical role in the pathology of me-v mice, as 7. O’Neill, E. M., I. Rebay, R. Tjian, and G. M. Rubin. 1994. 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grammed cell death. EMBO J. 11:3887. by guest on September 29, 2021 functions of Ets2 may be sufficiently complemented by Ets1, the 16. Fahy, R. J., A. I. Doseff, and M. D. Wewers. 1999. Spontaneous human monocyte most closely related Ets family member (M. Ostrowski and G. apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin Wei, unpublished observations), or by other signaling pathways. and is independent of caspase-1. J. Immunol. 163:1755. ␣ 17. Rozen, S., and H. Skaletsky. 2000. Primer3 on the WWW for general users and Genes such as Bcl-X and TNF- identified as Ets2 targets are for biologist programmers. In Bioinformatics Methods and Protocols: Methods in also targets for other transcription factors known to play important Molecular Biology. S. Krawetz and S. Misener, eds. Humana Press, Totowa, role in inflammatory responses, AP1 and NF-␬B (21, 22, 26). In- p. 365. ␬ 18. Shultz, L. D., P. A. Schweitzer, T. V. Rajan, T. Yi, J. N. Ihle, R. J. Matthews, terestingly, both AP1 and NF- B can directly interact with Ets2 or the M. L. Thomas, and D. R. Beier. 1993. Mutations at the murine motheaten locus highly related gene product Ets1 to activate target genes in many cell are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell types, including in the immune system (21, 22, 26). Studying genetic 73:1445. 19. Kovarik, J., L. Kuntz, B. Ryffel, and J. F. Borel. 1994. The viable motheaten interactions between Ets2 and these other transcription factors and (mev) mouse: a new model for arthritis. J. Autoimmun. 7:575. signaling pathways will provide a more detailed understanding of 20. Sementchenko, V. I., and D. K. Watson. 2000. Ets target genes: past, present and transcriptional regulatory networks controlling terminal differentiation future. Oncogene 19:6533. 21. Barthel, R., A. V. Tsytsykova, A. K. Barczak, E. Y. Tsai, C. C. Dascher, states in monocytes and macrophages, and may suggest new combi- M. B. Brenner, and A. E. Goldfeld. 2003. Regulation of tumor necrosis factor ␣ natory strategies to limit inflammation pathology involving these key gene expression by mycobacteria involves the assembly of a unique enhanceo- pathways. some dependent on the coactivator proteins CBP/p300. Mol. Cell. Biol. 23:526. 22. Sevilla, L., A. Zaldumbide, P. Pognonec, and K. E. Boulukos. 2001. Transcrip- tional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Acknowledgments Ets, Rel/NF␬B, STAT and AP1 transcription factor families. Histol. Histopathol. 16:595. We acknowledge Gustavo Leone and Cynthia Timmers for advice with 23. Thrall, R., S. Vogel, R. Evans, and L. Shultz. 1997. Role of tumor necrosis real-time PCR techniques, the Ohio State University Comprehensive Can- factor-␣ in the spontaneous development of pulmonary fibrosis in viable moth- cer Center Real-Time PCR Shared Resource, and the Keck Genetic Facility eaten mutant mice. Am. J. Pathol. 151:1303. for housing and care of mice. 24. Tsai, E. Y., J. V. Falvo, A. V. Tsytsykova, A. K. Barczak, A. M. Reimold, L. H. Glimcher, M. J. Fenton, D. C. Gordon, I. F. Dunn, and A. E. Goldfeld. 2000. A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, References and CREB binding protein and p300 is recruited to the tumor necrosis factor ␣ 1. Graves, B. J., and J. M. Petersen. 1998. Specificity within the ets family of promoter in vivo. Mol. Cell. Biol. 20:6084. transcription factors. Adv. Cancer Res. 75:1. 25. Yamamoto, H., M. L. Flannery, S. Kupriyanov, J. Pearce, S. R. McKercher, 2. Yang, B., C. Hauser, G. Henkel, M. Colman, C. Van Beveren, K. Stacey, G. W. Henkel, R. A. Maki, Z. Werb, and R. G. Oshima. 1998. Defective tro- D. Hume, R. Maki, and M. Ostrowski. 1996. Ras-mediated phosphorylation of a phoblast function in mice with a targeted mutation of Ets2. Genes Dev. 12:1315. conserved threonine residue enhances the transactivation activities of c-Ets1 and 26. Li, R., H. Pei, and D. K. Watson. 2000. Regulation of Ets function by protein- c-Ets2. Mol. Cell. Biol. 16:538. protein interactions. Oncogene 19:6514.