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Definitions

• the invention relates to animal models, in particular mouse models, for fibrotic diseases like pulmonary fibrosis and fibrotic disorders of the skin.
• Pulmonary fibrosis is a devastating disorder that affects five million people worldwide. However, the actual numbers may be significantly higher as a possible consequence of misdiagnosis. Typically, patients develop pulmonary fibrosis in their forties and fifties with symptoms that include shortness of breath, chronic cough, fatigue, loss of appetite and rapid weight loss. The mean survival time following diagnosis is less than 5 years (Giri, 2003). Pulmonary fibrosis is not seen as a separate entity but develops usually in the context of environmental exposures or as an accompaniment of a syndrome. Common causes are exposure to asbestos, metal dusts or organic substances, sarcoidosis (a disease characterized by the formation of granulomas), exposure to medical drugs and radiation. Often pulmonary fibrosis is associated with connective tissue or collagen diseases such as rheumatoid arthritis and scleroderma (Giri, 2003).
• the disease is characterized by chronic inflammation and collagen production within fibroblastic foci in the lung.
• Myofibroblasts a distinguishing feature of fibroblastic foci, are thought to arise from local activation of parenchymal fibroblasts by transforming growth factor ⁇ (TGF- ⁇ ) stimulation and are historically considered to be the collagen-producing cell in fibrotic lesions (Selman and Pardo, 2003); in addition, CTGF (connective tissue growth factor) is considered a very important factor and is required for differentiation and collagen gene expression.
• TGF- ⁇ transforming growth factor ⁇
• CTGF connective tissue growth factor
• the disease typically proceeds with scarring of the lung and the alveoli which become lined by fibrotic tissue. When the scar forms, the tissue becomes thicker causing an irreversible loss in efficiency of the tissue's ability to transfer oxygen into the bloodstream (Gross and Hunninghake, 2001).
• growth factors have been implicated in the pathogenesis of pulmonary fibrosis. These factors have been identified by virtue of their ability to stimulate fibroblast division and extracellular matrix (ECM) production, as well as their presence in the lungs and lung fluids of patients or animals with fibrotic lung disease. These growth factors include TGF- ⁇ , insulin-like growth factor (IGF)-I, platelet- derived growth factor (PDGF), members of the fibroblast growth factor (FGF) family and keratinocyte growth factor (KGF) (Krein and Winston, 2002).
• IGF insulin-like growth factor
• PDGF platelet- derived growth factor
• FGF fibroblast growth factor
• KGF keratinocyte growth factor
• pulmonary fibrosis is primarily caused by chronic inflammation (Gross and Hunninghake, 2001).
• fibrotic lung diseases are inflammatory disorders at their inception.
• pulmonary fibrosis develops in mice with ectopic expression of the inflammatory mediator tumor necrosis factor ⁇ (TNF- ⁇ ) in the lung (Miyazaki et al., 1995).
• TNF- ⁇ tumor necrosis factor ⁇
• a bleomycin model of pulmonary fibrosis in the mouse the fibrosis is preceded by profound inflammation, including the production of high levels of TNF- ⁇ (Piguet et al., 1989).
• TNF- ⁇ -deficient or TNF- ⁇ receptor-deficient mice are resistant against bleomycin-induced pulmonary fibrosis (Ortiz et al., 1998; Piguet et al., 1997). These results led to the assumption that fibrosis might be avoided when the inflammatory cascade is interrupted before irreversible tissue injury occurred. Thus, this theory explains the initial enthusiasm for corticosteroid and cytotoxic therapy of pulmonary fibrosis. However, it is now clear that the current antiinflammatory therapy provides little benefit (Giri, 2003). Therefore, some studies have attempted to show that fibrotic lung disorder is not an inflammatory disorder.
• fibrotic lung disease can be triggered by adenoviral transfer of TGF- ⁇ to the lungs of animals with only a transient inflammatory response.
• TGF- ⁇ results from sequential lung injury with a subsequent wound healing response rather than chronic injury. Therefore, a therapeutic strategy based on modification of fibroblast replication and matrix deposition is established.
• no beneficial clinical effect was seen in patients after colchicine treatment (interferes with intracellular collagen processing) or penicillamine treatment (collagen cross-link inhibitor).
• Other agents that are in experimental trials to block fibrogenesis include pirfenidone, interferon ⁇ or antibodies against TGF- ⁇ signaling (Giri, 2003).
• fibrotic disease e.g. pulmonary fibrosis that develops in the context of scleroderma-like disease (generalized fibrosis), or other fibrotic diseases.
• the solution of the problem underlying the invention is based on the molecular mechanisms associated with the transcription factor AP-I.
• the transcription factor AP-I is generated by a series of dimers of products of the Fos, Jun, and CREB/ATF protein families (Eferl and Wagner, 2003), as well as other bZip proteins.
• associations have been observed between Fos or Jun and the p65 subunit of NFKB (Stein et al., 1993), and ATF-2 and p50-NF ⁇ B (Du et al., 1993).
• Combinatorial association can draw on three Jun genes (c-jun, junB, junD), four Fos genes ⁇ c-fos, fosB, fra-1 , fra-2) and several CREB/ATF genes (Eferl and Wagner, 2003).
• mice lacking c-Fos develop osteopetrosis caused by a differentiation defect in the osteoclast lineage (Grigoriadis et al., 1994; Wang et al., 1992).
• This differentiation defect can be rescued by expression of the Fos-related protein Fra-1 suggesting that Fos and Fra-1 have overlapping functions in osteoclast differentiation (Fleischmann et al., 2000).
• Loss of Fra-1 in a conditional mouse model leads to reduced bone mass due to a functional defect of osteoblasts (Eferl et al., 2004).
• ectopic Fra-1 expression leads to osteosclerosis likely due to accelerated differentiation of osteoprogenitors into mature osteoblasts (Jochum et al., 2000).
• Ectopic expression designates the occurrence of gene expression in a tissue in which such gene is normally not expressed or normally expressed at a lower level.
• the genomic locus of Fra-2 was broadly overexpressed in a transgenic mouse (fra-2 tg ), originally with the purpose to investigate the consequence of increased Fra-2 activity on osteoblast and osteoclast functions.
• the transgenic vector for thGfra-2 tg mice was designed in a similar way as for c-fos tg and yr ⁇ -i tg mice (Grigoriadis et al., 1993; Jochum et al., 2000) to allow a meaningful comparison of the three transgenic mouse models.
• the invention relates to a an animal model for fibrotic diseases, comprising a non-human transgenic mammal, in particular a rodent, with broad or cell type-specific ectopic expression offra-2 that manifests itself in a phenotype of a fibrotic disease.
• fibrotic diseases relates to diseases involving fibrosis, which may e. g. be due to chronic inflammation or repair and reorganization of tissues. Fibrosis is the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to formation of fibrous tissue as a normal constituent of an organ or tissue, including the kidneys, heart, lungs, liver, skin and joints.
• the fibrotic disease is a pulmonary disease, in particular pulmonary fibrosis.
• pulmonary disease encompasses lung (pulmonary) fibrosis and pulmonary diseases with a fibrotic component selected from idiopathic pulmonary fibrosis, other interstitial pneumonias (IP) such as giant cell interstitial pneumonia, non-specific IP, cryptogenic organizing pneumonia, collagen vascular disease-associated IP, and drug-induced IP, also sarcodosis, cystic fibrosis, respiratory distress syndrome, granulomatosis, silicosis, asbestosis, systemic scleroderma involving the lung, as well as fibrosis and remodeling in asthma or COPD.
• IP interstitial pneumonias
• the fibrotic disease is generalized fibrosis, which is a skleroderma-like fibrosis that manifests itself in several target organs, including, without limitation, besides lung and/or skin, the spleen, heart, kidney and/or liver. It has also been shown in the experiments of the invention thatyr ⁇ -2 tg mice develop fibrotic disorders of the skin, which are associated with excessive healing. Keloids and hypertrophic scars in the skin are examples of such fibrotic skin disorders.
• animal model of the invention is also useful as a model for fibrotic skin disorders.
• transgenic rodent is meant a rodent (e.g., mouse, rat, hamster, etc.) having a non-endogenous (i.e., heterologous) nucleic acid sequence encoding Fra-2 DNA stably integrated into its germ line (i.e., in the genomic sequence of most or all of its cells).
• a. fra-2 transgene can be introduced into embryonic stem cells for generation of chimaeric mice with ectopic expression of fra-2.
• the rodent is a mouse.
• Heterologous Fra-2 nucleic acid is introduced into the germ line of such animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal according to standard protocols.
• UiQ fra-2 transgenic rodent/mouse is referred to as "fra-2 tg " rodent/mouse.
• fra-2 tg mouse or "fra-2 tg rodent”, respectively, in the meaning of the present invention, is a transgenic rodent/mouse carrying a fra-2 genomic locus o ⁇ fra-2 cDNA, under the control of a promoter that allows broad or cell-type specific transgene expression.
• fra-2 or “Fra-2” respectively
• r ⁇ -2 DNA or Fra-2 protein, respectively
• the chosenyr ⁇ -2 DNA may be identical to the homogenous fra-2 gene of the selected animal species (e.g.
• a mouse carries the murineyr ⁇ -2 DNA transgene and a rat the rat fra-2 transgene) or it may be different, e.g., in particular in view of the use of the animal as a model for human disease and for screening or characterizing inhibitors for human disease, it may be humanyr ⁇ -2.
• H2-K b promoter
• IRES-EGFP reporter gene
• LTR reporter gene
• enhancer the constituents H2-K b (promoter), IRES-EGFP (reporter gene) and LTR (enhancer) were used for the transgenic construct; these elements can be replaced by other elements, provided that expression of the transgene at the level and location in the body is such that it manifests itself in the phenotype of interest.
• ubiquitous promoters suitable for transgene expression are the ubiquitin C promotor, the CMV promoter/enhancer, the Pgk-1 promotor or the chicken ⁇ -actin promoter (Schorpp et al., 1996).
• reporter systems suitable for monitoring transgene expression are ⁇ -galactosidase, EGFP, EYFP, ERFP.
• An example for a suitable enhancer element that may be optionally present in the construct to ensure broad expression of fra-2 is the long terminal repeat (LTR) from FBJ murine osteosarcoma virus.
• a genomic mouse ⁇ DNA library (from mouse strain 129 Sv) was screened for the mouseyr ⁇ -2 gene with labeled oligonucleotides binding to the four exons o ⁇ fra-2.
• the fra-2 full- length locus was isolated, characterized and sequenced.
• the sequence of the four exons matched completely with the published mouse Fra-2 cDNA sequence (Foletta et al., 1994).
• the promoter for the major histocompatibility complex class I antigen H2-K b (Grigoriadis et al., 1993) was cloned in front of the fra-2 genomic locus to allow ubiquitous transgene expression.
• an IRES-EGFP (Zhu et al., 1999) sequence was cloned behind the/r ⁇ -2 locus followed by the long terminal repeat (LTR) sequence of the FBJ- murine sarcoma virus to stabilizeyr ⁇ -2 mRNA and to ensure transgene expression in mesenchymal cells (Grigoriadis et al., 1993).
• LTR long terminal repeat
• loxP sites were placed in front of exon 2 and after exon 4 to allow Cre-mediated deletion of transgene multimers and generation of several transgenic lines with different transgene copy numbers and levels of transgene expression.
• Theyr ⁇ -2 tg rodents in particular mice, can be obtained with methods based on known protocols for generating transgenic animals, e.g. by using the Cre-loxP mediated gene manipulation (as described e.g. by Orban et al., 1992; Sauer, 1993), by microinjection of fra-2 transgenes into fertilized oocytes, injection of large transgenes based on bacterial artificial chromosomes (BACs) into fertilized oocytes (as e.g.
• Cre-loxP mediated gene manipulation as described e.g. by Orban et al., 1992; Sauer, 1993
• BACs bacterial artificial chromosomes
• the present invention relates to a rodent, e.g. a mouse, with ectopic expression o ⁇ fra-2 in lung cells, e.g. airway epithelial cells, fibroblasts, myofibroblasts and hematopoietic cells, as an animal model for pulmonary fibrosis.
• a rodent e.g. a mouse
• lung cells e.g. airway epithelial cells, fibroblasts, myofibroblasts and hematopoietic cells
• cell-type specific promoters are used for generating the transgenic animal, e.g.
• surfactant protein C (Boggaram, 2003) promoter for airway epithelial cells, or the CD4 promoter (Tanigaki et al., 2004), the CD19 promoter (Rickert et al., 1997), the LysM promoter (Clausen et al., 1999) for hematopoietic cells or the pro-alpha 1(1) collagen promoter for fibroblasts (Rossert et al., 1995).
• ectopic expression may be desirable in other cells that are relevant for the phenotype of interest.
• yr ⁇ -2 expression can be achieved by directing expression of the transgene in these cells by using a cell-type specific promoter.
• the invention relates to transgenic rodent cells, in particular mouse cells, which have incorporated into their genome a human or rodentyr ⁇ -2 DNA.
• the fra-2 transgenic cells can be obtained from a rodent with ectopic expression of fra-2 either in a ubiquitous manner or specifically in the cell type of interest.
• the cells are selected with regard to their ability to contribute to development of the fibrotic disease, in particular pulmonary fibrosis, e.g. hematopoietic cells, pulmonary epithelial or mesenchymal cells like fibroblasts or myofibroblasts.
• fibroblasts and myofibroblasts are of particular interest.
• Cells of interest can be isolated and cultivated according to known methods, e.g. fibroblasts, myofibroblasts, lung epithelial cells (Dong et al., 1997; Phipps et al., 1989).
• the respective primary cells or cell lines can be transfected with/ra-2 DNA.
• the fra-2 tg mice and cells derived therefrom are useful for testing drugs for the treatment of fibrotic diseases like pulmonary disease.
• the animal model of the invention can also be used to test therapeutic strategies, e.g. compounds, for avoiding abnormal scar formation by pharmacological intervention.
• therapeutic strategies e.g. compounds
• fra-2 tg mice or fra-2 tg cells can be used in assays to screen for inhibitors of scar formation in excessive healing.
• the experiments of the invention have proven that Fra-2 itself is a target for pharmacological intervention of fibrotic disorders, including excessive scar formation. Therefore, Fra-2 inhibitors may be useful as inhibitors for such diseases.
• the animal model of the invention may be used, but is not limited to evaluate inhibitors of pro-inflammatory cytokines that play a role in fibrogenic pathways that depend on Fra-2 activity.
• novel inhibitors that might prevent microinjury of pulmonary epithelial cells such as angiotensin converting enzyme inhibitors or caspase inhibitors can be tested in the animal model of the invention.
• specific inhibitors that prevent the accumulation of collagen or in general the fibrogenic activity of cells in fibrotic foci of affected lungs can be tested.
• the transgenic animal and cell lines derived therefrom can also be used to identify or test candidate compounds effective in the therapy of fibrotic disease, in particular pulmonary fibrosis, e.g. by inhibiting not yet identified fibrogenic pathways that are dependent on Fra-2 activity.
• the present invention further provides a method of determining a compound's therapeutic effect on a fibrotic disease, e.g. pulmonary fibrosis.
• the method includes administering an effective amount of a test compound to afra-2 tg rodent, in particular a mouse.
• the method includes measuring the response of the transgenic animal to the test compound (i.e. the amelioration or disappearance of the disease symptoms), and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control.
• Compounds that may have an effect on the phenotype resulting from ectopic fra-2 expression may also be screened against cells in cell-based assays, for example, to identify such compounds.
• the compounds can be tested for their effect on excessive growth of fibroblasts and/or on transformation of fibroblasts to myofibroblasts and/or extracellular matrix formation.
• Toxicity and therapeutic efficacy of the identified compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
• Compounds that exhibit large therapeutic indices are preferred.
• While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. For example, achieving topical selectivity by way of inhaled compounds with short plasma half-lives to treat lung fibrosis.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. If the therapeutic window allows the use higher ED values without toxic effects, such ED values, e.g. up to ED 90, are preferred.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose may be formulated in an animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) or higher as determined in cell culture. (For agonists, values above the IC50 value (or the ED50 value for agonists) are preferred).
• Levels in plasma may be measured according to known methods, for example, by high performance liquid chromatography.
• the animal model is useful to study the etiology of human fibrotic diseases, e.g. pulmonary fibrosis. It is very important defining the cell type that is responsible for development of pulmonary fibrosis.
• the conventional bleomycin-induced pulmonary fibrosis models do not allow experiments such as bone marrow transplantation studies which hamper this goal.
• fra-2 tg mice can be bred into different genetic backgrounds such as mice with Rag2 deficiency or loss of TNF- ⁇ receptor p55 function to evaluate the contribution of autoreactive T-cells and TNF-signaling to pulmonary fibrosis, respectively.
• the knowledge about the responsible cell type and the major signaling pathways that can be obtained with the animal model according to the invention has a pivotal influence on the general direction of drug testing for treatment of pulmonary fibrosis.
• A. Schematic scheme of the fra-2 transgenic vector.
• An H2k b promoter is used for fra-2 expression.
• the IRES-EGFP reporter gene behindyr ⁇ -2 is used to monitor transgene activity and a
• LTR sequence is included for rnRNA stabilization.
• the loxP sites allow Cre-recombinase-mediated deletion of the transgene.
• H2K b H2Kb promoter
• E1-E4 Exon 1-4 o ⁇ fra-2
• polyA polyadenylation signal
• Hind III restriction sites used for Southern blot analysis.
• mice of line 12 and 13 show interstitial lung fibrosis (upper panel) and stenosis of blood vessels (lower panel).
• a scleroderma-like syndrome develops in fra-2 tg .
• Chrom- anilineblue-staining for collagen revealed that fibrotic lesions in fra-2 tg mice are not restricted to the lung but can also be observed in other organs (liver, skin and heart are shown as examples).
• Pulmonary fibrosis in fra-2 tg mice starts with vascular and perivascular immigration of inflammatory cells.
• H&E-staining of/r ⁇ -2 tg lungs demonstrates inflammation as the first event in pulmonary fibrosis (upper panel).
• Inflammatory cells are mainly composed of CD3 -positive T-cells (black staining, arrowheads) and esterase-positive myeloid cells such as granulocytes (red staining, arrowheads).
• Inflammation causes accumulation of chemokines and cytokines in the lungs of/r ⁇ -2 tg mice as demonstrated by RNase protection assay.
• Fra-2 transgenic mice show delayed wound closure after full-thickness wounding of the backskin (upper panel: 8 days after wounding, lower panel: 11 days after wounding).
• D. -G. Rnase protection assays with skin biopsies taken at wounding and 3 days after wounding. No difference in expression of AP-I members except for fra-2 (D), matrix metalloproteinases (E), TIMPs (E), cytokines (F), TGF-beta family members (F) and chemokines (G) can be observed.
• the fra-2 full-length locus is isolated from a genomic ⁇ DNA library, sequenced and cloned into the pBS II vector.
• the promoter for the major histocompatibility complex class I antigen H2-K b (Grigoriadis et al., 1993) is cloned in front of UiQ fra-2 genomic locus to allow ubiquitous transgene expression.
• an IRES-EGFP (Zhu et al., 1999) sequence is cloned behind the/ra-2 locus followed by the long terminal repeat (LTR) sequence of the FBJ-murine sarcoma virus (Grigoriadis et al., 1993).
• a loxP sites is placed in front of exon 2 and after exon 4 to allow Cre-mediated deletion of transgene multimers and generation of several transgenic lines with different transgene copy numbers.
• the transgenic construct is injected into the pronucleus of fertilized C57B176 oocytes and three independent transgenic lines are established.
• lO ⁇ g of tail DNA is digested with HindIII yielding a 12.5 kb fragment for the wild-typeyr ⁇ -2 allele and a 7.5 kb fragment for the transgene.
• HindIII For detection of the bands a 0.6 kb Kpnl fragment corresponding to exon 2 of fra-2 is used as probe.
• RNase protection assay total lung RNA is isolated with the TRIZOL protocol (Sigma). RNase protection assays are performed using the RiboQuant multi-probe RNase protection assay systems mCK-2b and mCK-5c (PharMingen) according to the manufacturer's protocol.
• Tissues are fixed overnight with neutral buffered 4% PFA at 4°C and embedded in paraffin.
• Five-micrometer sections are stained either with hematoxylin and eosin (HScE) or chromanilineblue or processed further.
• Immunohistochemical staining for anti CD3 is performed after antigen-retrieval (Dako S 1699) with the MultiLink Dako system (Dako E0453) according to the manufacturer's recommendations.
• Bone marrow is flushed out of femurs and tibias of donor mice, counted, and 5x10 6 cells are injected into the tail vein of recipients. Bone marrow and spleen cells from recipients were taken routinely after they were sacrificed for histological examination and the efficiency of reconstitution is evaluated by Southern Blot and FACS analysis for EGFP-fluorescence.
• the genomic fra-2 locus is placed on a transgenic vector with the broadly active H2K b promoter in front and a IRES-EGFP reporter gene behind (Fig. IA).
• a long terminal repeat (LTR) sequence of the FBJ-murine sarcoma virus is included for stabilization of fra-2 rnRNA and to ensure transgene expression in mesenchymal cells.
• LTR long terminal repeat
• the additional loxP sites that are placed in front of exon 2 and after exon 4 allow Cre-mediated deletion of transgene multimers and generation of several transgenic lines with different transgene copy numbers and levels of transgene expression. Three independent transgenic lines with different transgene copy numbers are generated (Fig. IB). Line 12 and 13 express the transgene at high levels (Fig.
• Line 15 carries only two transgene copies and did not show any overt transgene expression (Fig. 1C).
• transgene expression can be observed in all tissues of lines 12 and 13 except for the brain (Fig. ID).
• Pulmonary fibrosis is responsible for the premature lethality of the majority of/r ⁇ -2 tg mice (Fig. 2A). Mice become sick around 12 months of age and suffer from shortness of breath which is due to a dramatic increase in lung weight (Fig. 2B, 3A) and fibrosis of lung tissue (Fig. 3B). Pulmonary fibrosis often develops in response to environmental pollutants or in the context of a syndrome.
• Inflammation is one of the first events in pulmonary fibrosis
• Fig. 5A Immunohistochemical and histochemical stainings identify the major cell populations that infiltrate the lung. These are CD3 -positive T-cells and esterase-positive myeloid cells (Fig. 5A). The presence of these cells is associated with increased levels of chemokines and cytokines (Fig. 5B) which might contribute to the fibrogenic changes in the lung and might also attract more inflammatory cells.
• pulmonary fibrosis is primarily an autoimmune disease or if it is triggered by microinjury of airway epithelia with a more or less important contribution of the immune system.
• the disease should be transplantable withyr ⁇ -2 tg bone marrow in case thatyr ⁇ -2 tg mice develop autoreactive immune cells that are responsible for lung damage and pulmonary fibrosis.
• the disease should also develop in fra-2 tg mice that have been reconstituted with wild-type bone marrow cells in case that microinjury of airway epithelia is the primary trigger of the fibrosis.
• Fra-2 tg mice show delayed wound closure after full-thickness skin biopsy punch wounding of the backskin (Fig. 8A,C). An increased amount of granulation tissue consisting of collagen is visible in the wounds of/r ⁇ -2 tg mice after wounding (Fig. 8B). Rnase protection assays with skin biopsies taken at wounding and 3 days after wounding demonstrate no differences in expression of AP-I members (except for fra-2), matrix metalloproteinases, TIMPs, cytokines, TGF-beta family members and chemokines (Fig. 8D-G). These data demonstrate that increased Fra-2 activity leads to a delay in wound closure and to excessive scar formation. This suggests that inhibitors of Fra-2 activity can be applied ectopically to avoid scar formation after injury or surgery.
• Fra-1 replaces c-Fos-dependent functions in mice. Genes Dev 14, 2695-2700.
• Osteoblasts are target cells for transformation in c-fos transgenic mice. J Cell Biol 122, 685-701.
• c-Fos a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266, 443-448. Gross, T. J., and Hunninghake, G. W. (2001). Idiopathic pulmonary fibrosis. N Engl J Med 345, 517-525.
• Phipps R. P., Penney, D. P., Keng, P., Quill, H., Paxhia, A., Derdak, S., and Felch, M. E. (1989). Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC. Am J Respir Cell MoI Biol 1, 65-74.
• Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J Exp Med 170, 655-663.

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