Consequences of telomere dysfunction in fibroblasts, club and basal cells for lung fibrosis development.

TRF1 is an essential component of the telomeric protective complex or shelterin. We previously showed that dysfunctional telomeres in alveolar type II (ATII) cells lead to interstitial lung fibrosis. Here, we study the lung pathologies upon telomere dysfunction in fibroblasts, club and basal cells. TRF1 deficiency in lung fibroblasts, club and basal cells induced telomeric damage, proliferative defects, cell cycle arrest and apoptosis. While Trf1 deletion in fibroblasts does not spontaneously lead to lung pathologies, upon bleomycin challenge exacerbates lung fibrosis. Unlike in females, Trf1 deletion in club and basal cells from male mice resulted in lung inflammation and airway remodeling. Here, we show that depletion of TRF1 in fibroblasts, Club and basal cells does not lead to interstitial lung fibrosis, underscoring ATII cells as the relevant cell type for the origin of interstitial fibrosis. Our findings contribute to a better understanding of proper telomere protection in lung tissue homeostasis.

Targeting p16-induced senescence prevents cigarette smoke-induced emphysema by promoting IGF1/Akt1 signaling in mice.

Senescence is a mechanism associated with aging that alters tissue regeneration by depleting the stem cell pool. Chronic obstructive pulmonary disease (COPD) displays hallmarks of senescence, including a diminished stem cell population. DNA damage from cigarette smoke (CS) induces senescence via the p16 pathway. This study evaluated the contribution of p16 to CS-associated lung pathologies. p16 expression was prominent in human COPD lungs compared with normal subjects. CS induces impaired pulmonary function, emphysema, and increased alveolar epithelial cell (AECII) senescence in wild-type mice, whereas CS-exposed p16-/- mice exhibit normal pulmonary function, reduced emphysema, diminished AECII senescence, and increased pro-growth IGF1 signaling, suggesting that improved lung function in p16-/- mice was due to increased alveolar progenitor cell proliferation. In conclusion, our study suggests that targeting senescence may facilitate alveolar regeneration in COPD emphysema by promoting IGF1 proliferative signaling.

Identification of Optimal Mouse Models of Systemic Sclerosis by Interspecies Comparative Genomics.

OBJECTIVE: Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets. METHODS: Gene expression measured in skin from mice with sclerodermatous graft-versus-host disease (GVHD), bleomycin-induced fibrosis, Tsk1/+ or Tsk2/+ mice was mapped to human orthologs and compared to SSc skin biopsy-derived gene expression. Transforming growth factor ß (TGFß) activation was assessed using a responsive signature in mice, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression was measured in SSc patient and mouse skin. RESULTS: Gene expression in skin from mice with sclerodermatous GVHD and bleomycin-induced fibrosis corresponded to that in SSc patients in the inflammatory molecular subset. In contrast, Tsk2/+ mice showed gene expression corresponding to the fibroproliferative SSc subset. Enrichment of a TGFß-responsive signature was observed in both Tsk2/+ mice and mice with bleomycin-induced skin fibrosis. Expression of TNFRSF12A (the TWEAK receptor/fibroblast growth factor-inducible 14) was elevated in skin from patients with fibroproliferative SSc and the skin of Tsk2/+ mice. CONCLUSION: This study reveals similarities in cutaneous gene expression between distinct mouse models of SSc and specific molecular subsets of the disease. Different pathways underlie the intrinsic subsets including TGFß, interleukin-13 (IL-13), and IL-4. We identify a novel target, Tnfrsf12a, with elevated expression in skin from patients with fibroproliferative SSc and Tsk2/+ mice. These findings will inform mechanistic and translational preclinical studies in SSc.

Resolution of Skin Fibrosis by Neutralization of the Antifibrinolytic Function of Plasminogen Activator Inhibitor 1.

OBJECTIVE: Systemic sclerosis (SSc) is a fibrotic disease characterized by an obliterative vasculopathy with thrombosis and impairment of the coagulation-fibrinolysis balance. Plasminogen activator inhibitor 1 (PAI-1) is the major inhibitor of profibrinolytic plasminogen activators (PAs). This study was undertaken to evaluate the contribution of PAI-1 to SSc pathology in the skin. METHODS: PAI-1 was evaluated in skin from patients with diffuse SSc (dSSc) and those with limited SSc (lSSc) by immunohistochemistry. The contribution of PAI-1 to SSc pathology was tested in vivo in murine graft-versus-host disease (GVHD) and bleomycin models of progressive skin fibrosis and in vitro in dermal human microvascular endothelial cells (HMVECs) using a monoclonal antibody that selectively prevents the binding of PAI-1 to PA. RESULTS: Skin from patients with dSSc and those with lSSc showed increased PAI-1 levels in the epidermis and microvessel endothelium. PAI-1 neutralization in the GVHD model led to a dramatic, dose-dependent improvement in clinical skin score, concomitant with vasculopathy resolution, including a reduction in fibrinolysis regulators and vascular injury markers, as well as reduced inflammation. Resolution of vasculopathy and inflammation was associated with resolution of skin fibrosis, as assessed by reduction in collagen content and expression of key profibrotic mediators, including transforming growth factor ß1 and tissue inhibitor of metalloproteinases 1. Similar to the GVHD model, PAI-1 neutralization reduced dermal inflammation and fibrosis in the bleomycin model. PAI-1 neutralization stimulated plasmin-mediated metalloproteinase 1 activation in dermal HMVECs. CONCLUSION: Our findings indicate that neutralization of the antifibrinolytic function of PAI-1 resolves skin fibrosis by limiting the extent of initial vascular injury and connective tissue inflammation. These data suggest that PAI-1 represents an important checkpoint in disease pathology in human SSc.

Intrinsic gene expression subsets of diffuse cutaneous systemic sclerosis are stable in serial skin biopsies.

Skin biopsy gene expression was analyzed by DNA microarray from 13 diffuse cutaneous systemic sclerosis (dSSc) patients enrolled in an open-label study of rituximab, 9 dSSc patients not treated with rituximab, and 9 healthy controls. These data recapitulate the patient "intrinsic" gene expression subsets described previously, including fibroproliferative, inflammatory, and normal-like groups. Serial skin biopsies showed consistent and non-progressing gene expression over time, and importantly, the patients in the inflammatory subset do not move to the fibroproliferative subset, and vice versa. We were unable to detect significant differences in gene expression before and after rituximab treatment, consistent with an apparent lack of clinical response. Serial biopsies from each patient stayed within the same gene expression subset, regardless of treatment regimen or the time point at which they were taken. Collectively, these data emphasize the heterogeneous nature of SSc and demonstrate that the intrinsic subsets are an inherent, reproducible, and stable feature of the disease that is independent of disease duration. Moreover, these data have fundamental importance for the future development of personalized therapy for SSc; drugs targeting inflammation are likely to benefit those patients with an inflammatory signature, whereas drugs targeting fibrosis are likely to benefit those with a fibroproliferative signature.

Limited systemic sclerosis patients with pulmonary arterial hypertension show biomarkers of inflammation and vascular injury.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a common complication for individuals with limited systemic sclerosis (lSSc). The identification and characterization of biomarkers for lSSc-PAH should lead to less invasive screening, a better understanding of pathogenesis, and improved treatment. METHODS AND FINDINGS: Forty-nine PBMC samples were obtained from 21 lSSc subjects without PAH (lSSc-noPAH), 15 lSSc subjects with PAH (lSSc-PAH), and 10 healthy controls; three subjects provided PBMCs one year later. Genome-wide gene expression was measured for each sample. The levels of 89 cytokines were measured in serum from a subset of subjects by Multi-Analyte Profiling (MAP) immunoassays. Gene expression clearly distinguished lSSc samples from healthy controls, and separated lSSc-PAH from lSSc-NoPAH patients. Real-time quantitative PCR confirmed increased expression of 9 genes (ICAM1, IFNGR1, IL1B, IL13Ra1, JAK2, AIF1, CCR1, ALAS2, TIMP2) in lSSc-PAH patients. Increased circulating cytokine levels of inflammatory mediators such as TNF-alpha, IL1-beta, ICAM-1, and IL-6, and markers of vascular injury such as VCAM-1, VEGF, and von Willebrand Factor were found in lSSc-PAH subjects. CONCLUSIONS AND SIGNIFICANCE: The gene expression and cytokine profiles of lSSc-PAH patients suggest the presence of activated monocytes, and show markers of vascular injury and inflammation. These genes and factors could serve as biomarkers of PAH involvement in lSSc.

Antagonistic effect of the matricellular signaling protein CCN3 on TGF-beta- and Wnt-mediated fibrillinogenesis in systemic sclerosis and Marfan syndrome.

Abnormal fibrillinogenesis is associated with connective tissue disorders (CTDs), including Marfan syndrome (MFS), systemic sclerosis (SSc) and Tight-skin (Tsk) mice. We have previously shown that TGF-beta and Wnt stimulate fibrillin-1 assembly and that fibrillin-1 and the developmental regulator CCN3 are both highly increased in Tsk skin. We investigated the role of CCN3 in abnormal fibrillinogenesis in Tsk mice, MFS, and SSc. Smad3 deletion in Tsk mice decreased CCN3 overexpression, suggesting that TGF-beta mediates at least part of the effect of Tsk fibrillin on CCN3 which is consistent with a synergistic effect of TGF-beta and Wnt in vitro on CCN3 expression. Disruption of fibrillin-1 assembly by MFS fibrillin decreased CCN3 expression and skin from patients with early diffuse SSc showed a strong correlation between increased CCN3 and fibrillin-1 expression, suggesting that CCN3 regulation by fibrillin-1 extends to these CTDs. Diffuse SSc skin and sera also showed evidence of increased Wnt activity, implicating a Wnt stimulus behind this correlation. CCN3 overexpression markedly repressed fibrillin-1 assembly and also blocked other TGFbeta- and Wnt-regulated profibrotic gene expression. Together, these data indicate that CCN3 counter-regulates positive signals from TGF-beta and Wnt for fibrillin fibrillogenesis and profibrotic gene expression.

Increased expression of Wnt2 and SFRP4 in Tsk mouse skin: role of Wnt signaling in altered dermal fibrillin deposition and systemic sclerosis.

Systemic sclerosis (SSc) is a complex human disorder characterized by progressive skin fibrosis. To better understand the molecular basis of dermal fibrosis in SSc, we analyzed microarray gene expression in skin of the Tight-skin (Tsk) mouse, an animal model where skin fibrosis is caused by an in-frame duplication in fibrillin-1 (Fbn-1). Tsk skin showed increased mRNA levels of several genes involved in Wnt signaling, including Wnt2, Wnt9a, Wnt10b and Wnt11; Dapper homolog antagonist of beta-catenin (DACT1) and DACT2; Wnt-induced secreted protein 2; and secreted frizzled-related protein (SFRP)2 and SFRP4. RNase protection and northern blot confirmed microarray results. Furthermore, Wnt3a markedly stimulated matrix assembly of microfibrillar proteins, including Fbn-1, by cultured fibroblasts, suggesting that Wnts contribute to increased microfibrillar matrices in Tsk skin. Further analysis showed that SFRP4 expression is specifically increased in tissues expressing Tsk-Fbn-1, such as skeletal muscle and skin. The increase in SFRP4 mRNA in Tsk skin started 2 weeks after birth, following the increase in Wnt2 mRNA that occurred at birth. This suggests that SFRP4 may modulate Wnt functions in Tsk skin fibrosis. Lesional skin from SSc patients also showed large increases in SFRP4 mRNA and protein levels in the deep dermis compared to healthy skin, suggesting that the Wnt pathway might regulate skin fibrosis in SSc.

A macrophage marker, Siglec-1, is increased on circulating monocytes in patients with systemic sclerosis and induced by type I interferons and toll-like receptor agonists.

OBJECTIVE: Microarray analyses of peripheral blood leukocytes have shown that patients with systemic lupus erythematosus express increased levels of type I interferon (IFN)-regulated genes. In this study we examined gene expression by peripheral blood mononuclear cells (PBMCs) from patients with systemic sclerosis (SSc) to better understand the dysregulation of the immune system in this disease. METHODS: PBMC gene expression was analyzed by microarray and confirmed by real-time polymerase chain reaction (PCR). Surface protein expression of Siglec-1 was analyzed by flow cytometry in PBMCs from healthy control subjects and patients with SSc, and in control PBMCs that were cultured in vitro with Toll-like receptor (TLR) agonists. RESULTS: SSc patients showed increased expression of a cluster of IFN-regulated genes, including Siglec-1 (CD169, sialoadhesin). This result was verified and extended by real-time PCR, showing that a subset of the SSc patients expressed strikingly increased levels of Siglec-1 messenger RNA (mRNA). Flow cytometry of PBMCs from SSc patients and healthy controls showed increased Siglec-1 surface protein expression, which was restricted to CD14+ monocytes. In vitro studies showed that type I IFN and certain TLR agonists, including TLR-7 and TLR-9, induced Siglec-1 mRNA and protein expression. Moreover, TLR induction of surface Siglec-1 was shown to be type I IFN-dependent. Increased numbers of Siglec-1+ cells were observed by immunohistochemistry in the skin of SSc patients compared with healthy controls. CONCLUSION: Increased expression of Siglec-1 in circulating SSc monocytes and tissue macrophages suggests that type I IFN-mediated activation of monocytes occurs in SSc, possibly through TLR activation of IFN secretion. These observations indicate a potential role for type I IFN-activated monocyte/macrophages in the pathogenesis of SSc.

Transcription factor T-bet regulates skin sclerosis through its function in innate immunity and via IL-13.

Tissue remodeling with fibrosis is a predominant pathophysiological mechanism of many human diseases. Systemic sclerosis is a rare, often lethal, disorder of unknown etiology manifested by dermal fibrosis (scleroderma) and excessive connective tissue deposition in internal organs. Currently, there are no available antifibrotic therapeutics, a reflection of our lack of understanding of this process. Animal models of scleroderma are useful tools to dissect the transcription factors and cytokines that govern fibrosis. A disproportionate increase of type 2 cytokines, like TGF-beta and IL-4, more than type 1 cytokines, like IFN-gamma, is thought to underlie the pathogenesis of scleroderma. In this study, we show that mice deficient in the transcription factor T-box expressed in T cells (T-bet), a master regulator of type 1 immunity, display increased sensitivity to bleomycin-induced dermal sclerosis. Despite the well-established role of T-bet in adaptive immunity, we also show that RAG2(-/-) mice, which lack T and B cells, are vulnerable to bleomycin-induced scleroderma and that RAG2/T-bet double-deficient mice maintain the increased sensitivity to bleomycin observed in T-bet(-/-) mice. Furthermore, overexpression of T-bet in T cells does not affect the induction of skin sclerosis in this model. Lastly, we show that IL-13 is the profibrotic cytokine regulated by T-bet in this model. Together, we conclude that T-bet serves as a repressor of dermal sclerosis through an IL-13-dependent pathway in innate immune cells. T-bet, and its transcriptional network, represent an attractive target for the treatment of systemic sclerosis and other fibrosing disorders.

Microfibril-associated MAGP-2 stimulates elastic fiber assembly.

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.

Fibrillin in Marfan syndrome and tight skin mice provides new insights into transforming growth factor-beta regulation and systemic sclerosis.

PURPOSE OF REVIEW: Important recent understandings of fibrillins and fibrillin-associated microfibril proteins suggest new ways these proteins might contribute to tissue fibrosis seen in systemic sclerosis by regulating latent transforming growth factor-beta. This review discusses mutant-fibrillin mouse models of Marfan syndrome and SSc (Tsk mice), and studies suggesting that alterations in microfibrils might contribute to human SSc. RECENT FINDINGS: Fibrillin-1 mutations associated with Marfan syndrome have recently been shown to induce genes activated by TGF-beta. The inhibition of TGF-beta in these mouse models largely reverses phenotypic and pathologic disease manifestations. Recent studies suggest that alterations in the fibrillin-1 structure from mutant Tsk fibrillin cause hypodermal fibrosis and associated changes in dermal gene expression, suggesting stimulation of cytokine-mediating signals. Genetic mutations in fibrillin-1, in a higher frequency in SSc patient populations, and autoantibodies to fibrillin provide potential links to human SSc. SUMMARY: Fibrillin is placed centrally not only as the primary structural component of microfibrils, but also a key regulator of cytokines in the TGF-beta superfamily. Fibrillin may thus communicate alterations in matrix to fibroblast gene expression. These observations complement emerging understandings of the effects of Tsk fibrillin, and genetic and autoimmune studies of human fibrillin on dermal fibrosis.

Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts.

Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.

Increased expression of type I collagen induced by microfibril-associated glycoprotein 2: novel mechanistic insights into the molecular basis of dermal fibrosis in scleroderma.

OBJECTIVE: Mutations in fibrillin 1, a key component of extracellular microfibrils, are associated with connective tissue disorders such as Marfan's syndrome or skin fibrosis in the tight skin mouse model of scleroderma. Previous studies have suggested that fibrillin 1 mediates skin fibrosis via its interface with associated microfibrillar proteins and type I collagen; in particular, microfibril-associated glycoprotein 2 (MAGP-2), an extracellular matrix protein that binds to fibrillins and the alphavbeta3 integrin, is increased in TSK mouse and human scleroderma skin. Because the function of MAGP-2 in the biologic processes of the matrix remains unknown, this study investigated whether MAGP-2 regulates type I collagen. METHODS: Fibroblast cultures conditionally overexpressing MAGP-2 were developed. Cells were analyzed by Western blotting, Northern blotting, pulse-chase analysis, and immunofluorescence to assess the effect of MAGP-2 on type I collagen. RESULTS: Cells overexpressing MAGP-2 formed increased MAGP-2 matrix and showed a 3-fold increase in intracellular type I procollagen. This increase was associated with increased levels of type I collagen in the medium and matrix. Increased type I collagen colocalized with the MAGP-2 matrix. MAGP-2 overexpression had no effect on type I procollagen messenger RNA, but markedly increased the half-life of type I procollagen. MAGP-2 induced type I collagen even under conditions in which no MAGP-2 matrix was detectable, and did not require the presence of the RGD motif of MAGP-2 in its integrin-binding site. CONCLUSION: This study shows that MAGP-2 stabilizes type I procollagen, identifying an important function of MAGP-2 in extracellular matrix homeostasis. It also suggests that MAGP-2 might mediate skin fibrosis in TSK mice and in patients with scleroderma.

Fibulin-2 and fibulin-5 alterations in tsk mice associated with disorganized hypodermal elastic fibers and skin tethering.

The Tight skin (Tsk) mouse is an important model of skin fibrosis that occurs in systemic sclerosis. These mice develop skin tethering and thickening associated with expression of a mutant fibrillin-1 gene. We show that Tsk fibrillin-1 leads to marked alterations in elastic fibers of the hypodermis of Tsk animals. In Tsk mice, a prominent elastic fiber layer found normally at the interface between hypodermal muscle and connective tissue was absent from an early age. The lack of elastic fibers at the hypodermal muscle-connective tissue (M-CT) interface was associated with a loss of staining for fibulin-5 in the same region. These mice also formed disorganized elastic fibers throughout hypodermal connective tissue as they aged. The increased elastic fibers in Tsk hypodermal connective tissue was associated with increased fibrillin-1 and fibulin-2 matrices. These results suggest that Tsk fibrillin-1 causes skin tethering by altering matrix protein composition in Tsk hypodermal connective tissues. The closely parallel alterations in elastogenesis associated with increased fibulin-2 in hypodermal connective tissues and decreased fibulin-5 at the hypodermal M-CT interface suggest that these proteins mediate the effect of Tsk-fibrillin-1 on elastogenesis.

Mutant fibrillin 1 from tight skin mice increases extracellular matrix incorporation of microfibril-associated glycoprotein 2 and type I collagen.

OBJECTIVE: Skin fibrosis in the TSK mouse, a model of skin fibrosis seen in systemic sclerosis (SSc), is caused by a large in-frame duplication in the Fbn1 gene, tsk-Fbn1. We investigated whether tsk-Fbn1 might cause dermal fibrosis by affecting Fbn1 and associated extracellular matrices. We also studied whether deposition of microfibril-associated glycoprotein 2 (MAGP-2), a protein that is associated with fibrillin 1, was altered in the skin of patients with SSc. METHODS: An in vitro model of the TSK mouse was created by conditionally expressing tsk-Fbn1 in mouse embryonic fibroblasts (MEFs). Cell cultures were examined by immunofluorescence and Western and Northern blotting to determine the effect of tsk-Fbn1 on the structure, expression, and deposition of fibrillin 1 (Fbn-1), type I collagen, and MAGP-2. The skin of TSK mice and SSc patients was analyzed by immunohistochemistry for MAGP-2 expression. RESULTS: Expression of tsk-Fbn1 in cultured MEF cells altered the morphology of Fbn-1 fibers and increased the deposition of type I collagen into the extracellular matrix (ECM) without concomitantly changing messenger RNA expression, secretion, or processing of type I procollagen. Moreover, MEF cells expressing tsk-Fbn1 showed increased MAGP-2 matrix. MAGP-2 was increased in the dermis of TSK mice. Fibrotic SSc skin also showed higher levels of MAGP-2 in the dermis than nonfibrotic SSc skin and normal skin. CONCLUSION: Tsk-Fbn1 altered ECM organization and caused fibrosis by affecting the deposition of MAGP-2 or other Fbn-1-associated proteins. Alterations in microfibril structure or deposition might contribute to fibrosis in SSc.

Transforming growth factor beta induces fibroblast fibrillin-1 matrix formation.

OBJECTIVE: Fibrillin, an extracellular matrix protein implicated in dermal fibrosis, is increased in the reticular dermis of systemic sclerosis (SSc) skin. We undertook this study to investigate the hypothesis that transforming growth factor beta (TGFbeta) or other cytokines regulate fibrillin matrix formation by normal and SSc fibroblasts. We further investigated the mechanism of TGFbeta-induced fibrillin fibrillogenesis and its relationship to myofibroblasts. METHODS: Fibrillin and fibronectin matrix deposition and alpha-smooth muscle actin expression by fibroblast cultures from normal and SSc skin treated with TGFbeta or other cytokines were analyzed by immunofluorescence. Supernatant and extracellular matrix from normal and SSc fibroblasts treated with or without TGFbeta were evaluated by Western blot and Northern blot for fibrillin protein and messenger RNA (mRNA) expression, respectively. RESULTS: Immunofluorescence demonstrated increased fibrillin matrix formation by normal and scleroderma fibroblasts after TGFbeta treatment. Other cytokines, including tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-4, granulocyte-macrophage colony-stimulating factor, and platelet-derived growth factor, did not affect fibrillin fibrillogenesis. Fibrillin matrix formed in proximity to myofibroblasts and independently of up-regulation of fibronectin matrix or cell number. Western blot analysis of extracellular matrix confirmed increased fibrillin after TGFbeta stimulation of normal or scleroderma fibroblasts. However, TGFbeta did not alter the expression of either soluble fibrillin protein or fibrillin mRNA. CONCLUSION: Our data show that TGFbeta induces fibrillin protein incorporation into the extracellular matrix without affecting fibrillin gene expression or protein synthesis, suggesting that fibrillin matrix assembly is regulated extracellularly. TGFbeta might increase fibrillin matrix by activating myofibroblasts. Such TGFbeta-mediated effects could account for the increased fibrillin matrix observed in SSc skin.

Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2: a novel function for SR proteins.

Pre-mRNA splicing is a widely used regulatory mechanism for controlling gene expression, and a family of conserved proteins, SR proteins, participate in both constitutive and alternative splicing. Here we describe a novel function for the SR protein ASF/SF2. We used an embryonic chicken cDNA library to screen for differential mRNA expression in the chicken B-cell line DT40-ASF, expressing or not expressing ASF/SF2. Remarkably, out of 3 x 10(6) clones screened, only one, isolated several times independently, showed ASF/SF2-related differential expression. The isolated cDNA, referred to here as PKCI-r (for PKCI-related), is closely related to the protein kinase C interacting protein (PKCI-1) gene. Transcript levels were increased approximately sixfold in ASF/SF2-depleted cells compared with cells expressing ASF/SF2, indicating a negative role for the SR protein. Strikingly, inhibition of ASF/SF2 expression had no significant effect on PKCI-r splicing, or transcription, but markedly increased the half-life of PKCI-r mRNA (6.6-fold). Similarly, increased mRNA stability was also observed upon expression of exogenous PKCI-r mRNA in cells depleted of ASF/SF2. ASF/SF2 bound to a discrete region containing a purine-rich sequence in the 3' UTR of the PKCI-r transcript, and deletion of this region eliminated ASF/SF2-mediated regulation of transcript stability. Together these data indicate a novel, direct effect of ASF/SF2 on PKCI-r mRNA stability. Therefore, ASF/SF2, and perhaps other SR proteins, affects gene expression in vertebrate cells through regulation of mRNA stability as well as splicing.