This study supports a novel and important role for the phosphorylation of threonine-72 of ets-2 in human and murine pulmonary fibrosis. These studies were performed in response to observations by Trimboli and colleagues (13
) and Wei and colleagues (18
), who found that mutating the threonine-72 to alanine-72 of ets-2 in mice altered lung inflammation and extracellular matrix remodeling, two important mechanisms in the pathogenesis of pulmonary fibrosis.
The consensus core binding sequence for ets-2 is 5′-GGAA/T-3′ (27). Upon binding this core sequence, ets-2 supports a diversity of cellular functions, such as cell proliferation, adhesion, migration, survival, and angiogenic processes (27
). As such, ets-2 plays an important role in several human diseases, including mammary tumors (13
), Down syndrome (28
), and leukemia (29
). Although ets-2 is central in a host of processes, pinpointing the sole genetic target responsible for disease manifestations as a result of dysregulated ets-2 function is difficult. For example, in the original study of lung inflammation using ets-2 (A72/A72) mice, Wei and colleagues found that 10 different genes were down-regulated in the lungs of ets-2 (A72/A72) mice compared with ets-2 (WT/WT) mice, but they did not identify a single dominant ets-2–responsive gene responsible for the decreased inflammation (18
). The report by Wei and colleagues indicates that ets-2 contributes to decreased lung inflammation by affecting genes with ets-2 promoter sites, supporting the notion that ets-2 is a multitarget transcription factor involved in numerous biological processes.
The promoter-binding capabilities and functions of the mutant form of ets-2 (ets-2 [A72]) are noteworthy. Previous studies demonstrated that the ets-2 (A72) protein translocates to the nucleus and binds a rat sarcoma (Ras)-responsive enhancer without activating the gene (16
). As such, the mutant form of ets-2 may serve as a co-repressor. For example, the BS69 protein functions as a transcriptional co-repressor when associated with E1A and ets-2 (30
). In the absence of ets-2, BS69 may not function as a co-repressor of fibrotic gene expression. Interestingly, the association of BS69 and ets-2 is decreased when ets-2 is phosphorylated at the pointed domain, which contains the threonine-72 residue (31
). Although several other co-repressors are thought to function coordinately with ets-2, including brahma-related gene 1 and the switch/sucrose nonfermentable (SWI/SNF) complexes (32
), the mechanisms by which altered genetic co-repression contributes to the pathogenesis of pulmonary fibrosis require further study.
Three additional findings in this study concern the involvement of ets-2 in the expression of C-C chemokine ligand 12 (CCL12) and differences in lung inflammatory cell differentials and primary cell proliferative capacities between ets-2 (WT/WT) and ets-2 (A72/A72) mice. First, using bone marrow–derived macrophages stimulated with M-CSF, we observed that macrophages from ets-2 (A72/A72) mice expressed significantly less CCL12, as detected via ELISA, than macrophages from ets-2 (WT/WT) mice (Figure E3A). Furthermore, we observed that ets-2 (A72/A72) mice had significantly less CCL12 in the lungs after treatment with bleomycin, as detected via ELISA and real-time PCR (Figures E3B and E3C).
Second, as shown in Table E1, BAL fluid cell differentials revealed that ets-2 (A72/A72) mice manifested significantly fewer total cells and alveolar macrophages 11 days after the initiation of bleomycin, compared with ets-2 (WT/WT) mice. Interestingly, this trend reversed at 22 days after the initiation of bleomycin, insofar as ets-2 (A72/A72) mice exhibited a significantly increased number of total cells and alveolar macrophages compared with ets-2 (WT/WT) mice. By 33 days after the initiation of bleomycin, no significant difference in total cells or alveolar macrophages was evident between ets-2 (WT/WT) and ets-2 (A72/A72) mice after treatment with bleomycin.
Third, fibroblasts isolated from human fibrotic lungs exhibit variability in proliferative and apoptotic capacities that may contribute to the pathogenesis of the disease (33
). Interestingly, primary lung fibroblasts (Figure E4) isolated from ets-2 (A72/A72) mice showed an increased proliferative capacity when compared with those cells isolated from ets-2 (WT/WT) mice. Although more studies are needed, our findings that ets-2 is a potential regulator of CCL12 expression, influences inflammatory cell profiles in the lungs in response to bleomycin, and exerts an effect on cell-proliferative capacities provide valuable insights into the importance of ets-2 in pulmonary fibrosis.
Recent studies highlighted the important role of transcription factors in the pathogenesis of pulmonary fibrosis by regulating fibrotic gene expression. For example, Lepparanta and colleagues demonstrated that the expression of GATA-6 in fibroblasts mediates the α-SMA–inducing signal of TGF-β, and that GATA-6 is overexpressed in the fibroblastic foci of lung sections from patients with IPF (36
). Yasuoka and colleagues ascertained that the expression of early growth response 1 is up-regulated in lung sections from patients with IPF, and directs the expression of fibronectin in response to the stimulation of fibroblasts by (insulin-like growth factor binding protein 5) (37
). Ponticos and colleagues highlighted the relevance of the mitogen-activated protein kinase (MAPK) pathway in the transcriptional activation of Type I collagen (38
), to which ets-2 is implicitly related. Furthermore, Boon and colleagues demonstrated that genes in the extracellular signal-related kinase/MAPK pathway are up-regulated in patients with IPF, and these genes include a member of the SWI/SNF family and Ras (39
Ets-2 can be activated by multiple signaling pathways, including Ras–v-raf-1 murine leukemia viral oncogene homolog (Raf)–MAPK (16
), PI3-kinase/mammalian target of rapamycin (40
), and v-akt murine thymoma viral oncogene homolog/c-Jun N-terminal kinase (41
). Raf is also activated via Ras-independent mechanisms (42
), thereby creating a complex network of intracellular signaling mechanisms that facilitate the activation of ets-2. In the setting of IPF, broad pathway inhibitors have been used, such as cytotoxic agents and immune suppressants, but none were effective in reversing the course of the disease or slowing its progression. This raises the possibility of novel and specific therapeutic approaches for IPF, centering on the promoter-binding inhibitors of ets-2–mediated transcription or the direct targeting of phosphorylated ets-2. This more directed, specific therapeutic approach was successfully applied in other diseases, including breast cancer (tamoxifen) and rheumatoid arthritis (etanercept), and should be considered in the treatment of IPF. Ets-2 antagonists or inhibitors of phosphorylated ets-2 are presently unavailable; we are actively pursuing their development and use.