Scleroderma, which is also known as systemic sclerosis (SSc), is a multisystem connective tissue disorder in humans that leads to fibrosis of the skin and internal organs and often results in death (1, 2). The etiology of SSc remains incompletely understood (3). Early pathologic hallmarks of the disease include immunologic activation associated with the development of a range of autoantibodies, microvascular endothelial cell activation, and later perivascular infiltration of mononuclear immune cells (4). As a consequence of these early events, fibroblasts within the skin and affected internal organs are activated, leading to excessive deposition of extracellular matrix (ECM) components (4). A proportion of fibroblasts differentiate into myofibroblasts, which are believed to be central to the development of fibrosis (5–7). As the disease becomes established, the skin becomes fibrotic and small blood vessel walls thicken. Eventually, the lungs, heart, kidneys, and gastrointestinal tract are involved, but there is substantial heterogeneity in the morbidity and mortality due to SSc (3). Secondary events, including exposure to environmental agents, hemodynamic stress, or epithelial injury, may trigger involvement of target organs. The disease can therefore be characterized as ubiquitous fibrosis of the skin and vasculature, together with increased susceptibility to more widespread organ-specific fibrosis.
There is considerable evidence implicating overactivity of the pleiotropic growth factor transforming growth factor β (TGFβ) in the pathogenesis of SSc (8, 9). Thus, elevated levels of TGFβ have been reported in SSc, and elevated expression of TGFβ protein or messenger RNA in the skin and lung has been reported (10). In addition, the altered profile of gene and protein expression characteristic of SSc fibroblasts is reminiscent of that of normal fibroblasts activated by TGFβ (11–13). It has therefore been hypothesized that TGFβ released from a variety of cell types activates fibroblasts in SSc. Testing this hypothesis experimentally is difficult owing to the pleiotropic effects of TGFβ on multiple cell types, its key role in embryonic development, and the complexity of TGFβ regulatory mechanisms in vivo.
Because of the proposed central role of TGFβ in SSc (8, 9) and the involvement of fibroblasts and fibroblast-like cells in the disease, we postulated that a transgenic mouse model in which TGFβ signaling in these cells is increased would recapitulate many key features of the disease. Investigators in our group had previously identified a strong transcriptional enhancer located between 15 kb and 19 kb upstream of the transcription start site of the Col1a2 gene that was largely fibroblast specific (14), so we attempted to generate transgenic mice in which this enhancer directed the expression of constitutively active type I TGFβ receptor (TGFβRI). Unfortunately, all embryos died early during gestation, presumably because there was a disruption of TGFβ signaling that caused embryonic death (data not shown). To circumvent this, we used the Cre/loxP system in which tamoxifen-inducible recombinase can be activated after birth. We were able to produce a mouse in which TGFβ signaling was disrupted only after the developmental progress of the embryo had been largely completed. We report here that the expression of constitutively active TGFβRI after birth in fibroblastic cells from these mice caused generalized skin fibrosis and a thickening of the walls of the small arteries, reproducing 2 of the hallmarks of human SSc.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we have demonstrated that the TBRICA; Cre-ER mice recapitulate key pathologic features of SSc when they are injected with 4-OHT, which activated TGFβ signaling. These features included a marked skin fibrosis that increased with age and a thickening of the walls of small arteries in lung and kidney. In vivo and in vitro measurements of collagen indicated a marked increase in collagen accumulation and collagen gene expression. Our results are consistent with a central role of TGFβ in disease pathogenesis (8, 9), and they identify the fibroblast as a critical target cell (11–13). A logical implication of this view is that other pathogenic processes such as immunologic activation or endothelial cell dysfunction, both of which are observed early in scleroderma, lead to TGFβ-mediated activation of fibroblasts in vivo. The conditional activation of signaling using the Cre/loxP system with deletion of a transcription stop cassette is a powerful method, since it allows high-level activation that is transmissible to daughter cells at mitosis and does not depend upon the continued presence of the tamoxifen ligand.
Explanted dermal fibroblasts showed many of the hallmark biochemical properties of SSc. Key features included myofibroblast differentiation, ECM overproduction, and constitutive overexpression of a number of downstream TGFβ targets. Two TGFβ-responsive Smad-dependent promoters were also up-regulated in the mutant fibroblasts. We therefore hypothesize that the activation of the genes observed in the mutant fibroblasts is mediated by the constitutive activity of the Smad pathway. As in fibroblasts from SSc patients (31), activation of the MAPK and p38 pathways in the fibroblasts of mutant mice was also observed.
The mutant mice did not show fibrotic lesions in the lungs or renal insufficiency, which are often late manifestations of human scleroderma. One possible explanation is that by bypassing an initial inflammatory component in our mouse model, we have eliminated a critical initiating event that leads to the lung pathology seen in scleroderma. Additionally, our mice are housed in a specific pathogen–free facility and are completely free of any pathogenic agents, while in the real world, exposure to such agents might be a possible injury or trigger in initiating the cascade of events that culminate in the inflammatory/autoimmune features of scleroderma. We have, however, recently observed that intratracheal administration of bleomycin in the same 4-OHT–treated mutant mice causes a more pronounced fibrotic response in the lungs than in control mice (Sonnylal S: unpublished observations). It is possible that additional injuries are needed to trigger renal insufficiency in TBR1CA; Cre-ER mice.
In fibroblasts derived from mutant mice, there was a marked increase in the levels of CTGF RNA. Similar increases have been seen in fibroblasts from scleroderma patients and after TGFβ stimulation of normal fibroblasts (26). Like TGFβ, CTGF can induce matrix production in fibroblasts (32, 33). We have recently generated transgenic mice that overexpress CTGF in fibroblastic cells. These mice also show generalized skin fibrosis (Sonnylal S: unpublished observations). Comparison of the changes in specific RNA levels and of the activities of specific signaling pathways in the fibroblasts of these mice with those in which constitutively active TGFβRI is expressed in fibroblasts should aid in understanding the mechanisms of CTGF function.
During wound healing, TGFβ and other cytokines are known to promote ECM production. Any disruption or excessive activity of these signals can result either in impairment or in excess tissue formation, resulting in fibrosis. In tissue fibrosis, the fine balance that exists between the expression of matrix metalloproteinases and the expression of TIMPs is impaired (34). Studies investigating the association of TIMPs with fibrosis in scleroderma have found elevation of TIMP-1 levels increasing with the severity of the disease (35). The increased levels of TIMP-1 expression in fibroblasts that express constitutively active TGFβRI are likely to have contributed substantially to the fibrotic phenotype of our mutant mice.
Animal models have been very valuable in defining pathogenic mechanisms as well as testing therapeutic interventions in human disease. Although there are several available models of SSc, each of these incompletely represents the human condition (17, 36–40). For example, the tight skin mouse develops dermal fibrosis, but the pathogenic mechanisms are not defined (36). In addition, recent reexamination of these mice has challenged their usefulness as a model of scleroderma (41). Subcutaneous injection of bleomycin induces transient skin sclerosis only in animals with a susceptible genetic background, and this diminishes once the injurious stimulus is removed (39). Minimal mismatch graft-versus-host disease has been used to investigate the potential for TGFβ-blocking therapies but has proven variable in severity and is generally self-limiting (40).
The mice with the constitutively active fibroblast-specific TGFβRI develop 2 cardinal features of established SSc, dermal fibrosis and structural vasculopathy. In addition, there is evidence of generalized abnormalities in ECM deposition extending beyond sites that are clinically involved, similar to what is now well recognized in SSc. This mouse model offers substantial advantages that include a defined nature, the primary defect being activation of TGFβ signaling in fibroblasts. Our mouse model can be regulated—this allows the developmental consequences during embryogenesis of altered TGFβ signaling to be avoided. While we appreciate that the potentially critical early immunologic and inflammatory components of human SSc are not reproduced in this model, our findings provide important direct evidence that activating TGFβ signaling in just the fibroblastic lineage replicates key fibrotic features of SSc. This suggests that in vivo in human disease, one of the eventual consequences of these (vascular, immunologic) events is activation of TGFβ signaling in fibroblasts.
By circumventing these events, our model is uniquely placed to study the late-stage fibrotic phase of SSc, which is particularly difficult to treat. It allows long-term sustained pathway activation, and so, overcomes some of the limitations of induced models. This model also offers substantial potential for the evaluation of therapies that target TGFβ activation or downstream consequences of this activation, including the effects of secondary mediators such as CTGF and others. The ability to induce the disease phenotype at defined time points will allow prevention as well as treatment strategies to be examined. These mice will allow other injurious stimuli to be evaluated, including vascular, immunologic, or pulmonary epithelial injury. For example, epithelial abnormalities may be important, and these may have different effects during development and during the postnatal period. These mice show much-increased susceptibility to bleomycin-induced lung disease (results not shown), providing strong validation of our model in SSc.
In conclusion, the characterization of a novel mouse model, in which TGFβ signaling can be activated selectively in fibroblasts at defined postnatal time points, provides strong direct evidence of a pivotal role of this growth factor in fibrotic diseases and scleroderma. Future modifications of this model are likely to result in an even more comprehensive clinical scleroderma phenotype. Our model, which provides insight into pathogenic mechanisms in this disease, will also be used as a platform to evaluate therapies targeting TGFβ activation.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. de Crombrugghe had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Drs. Sonnylal, Denton, Zheng, Behringer, and de Crombrugghe.
Acquisition of data. Drs. Sonnylal, Denton, Zheng, Keene, and He, Mr. Adams, and Ms Deng.
Analysis and interpretation of data. Drs. Sonnylal, Denton, Keene, VanPelt, Geng, and de Crombrugghe.
Manuscript preparation. Drs. Sonnylal, Denton, and de Crombrugghe.
Statistical analysis. Dr. Sonnylal.