Latent TGF-beta Binding Protein Research Articles

Local TGF-beta sequestration by fibrillin-1 regulates vascular wall homeostasis in the thoracic aorta

Abstract Background Aortic dissection and rupture is the main cause of early cardiovascular mortality in patients with Marfan syndrome (MFS). MFS is caused by a defect in fibrillin-1, a building block of microfibrils in the extracellular matrix which binds transforming growth factor beta (TGF-beta) via interaction with latent TGF-beta binding proteins (LTBPs). Multiple mouse models, both pharmaceutically induced and genetically manipulated, have been used to investigate the pathophysiology and biomechanical aspects of thoracic aortic aneurysms and dissections. However, the role of TGF-beta in MFS has been controversial, with earlier studies suggesting that excess release of TGF-beta due to decreased interaction with dysfunctional fibrillin-1 leads to aortic dilation and vascular damage, while other studies have shown an important protective effect of TGF-beta. Studying dedicated mouse models for MFS, with defects interfering with TGF-beta binding and -function may help resolve these discrepancies. Purpose This study aimed to reveal insights in the role of TGF-beta signaling in aneurysm formation and dissection in MFS. Methods Mice lacking the fibrillin-1 binding site for LTBPs (Fbn1H1Δ/+ and Fbn1H1Δ/H1Δ), mice with a truncated fibrillin-1 (Fbn1GT-8/+), and mice with a combination of both alleles (Fbn1GT-8/ H1Δ) were subjected to in vivo cardiac ultrasound analysis. Ex vivo phase-contrast synchrotron X-ray imaging was performed at the Paul Scherrer Institute to visualize the elastic lamellae architecture in the vascular wall of the entire excised thoracic aorta in a subset of mice from each group. Results Fbn1GT-8/+, Fbn1 H1Δ/+ and Fbn1H1Δ/H1Δ mice had a normal life span, but Fbn1GT-8/ H1Δ mice showed increased mortality due to aortic rupture starting at 4–5 months of age. The aortic root was dilated both in Fbn1GT-8/+ and Fbn1GT-8/ H1Δ mice at 6 months of age, but not in Fbn1H1D/+ or Fbn1H1Δ/H1Δ mice. Synchrotron images showed significant elastic lamellae fragmentation in the thoracic aortic wall of Fbn1GT-8/+ mice, and to a larger extent in Fbn1GT-8/ H1Δ mice. Surprisingly, localized elastin fragmentation was also found in the ascending thoracic aorta of Fbn1 H1Δ/+ and Fbn1H1Δ/H1Δ mice, despite a lack of aortic aneurysm formation. Moreover, Fbn1H1Δ/H1Δ mice displayed more severe aortic wall damage. The localized microdissections found in these mouse models were characterized by a severe alteration of the elastic fiber organization, cellular influx and increased collagen deposition, as confirmed by histological analysis. Conclusions Our data suggest that loss of LTBP binding to fibrillin-1 leads to the development of localized microdissections in the aorta in the absence of aortic aneurysm, and exacerbates the aortic wall morphology abnormalities in mice with truncated fibrillin-1. We therefore hypothesize that local TGF-beta sequestration is required to maintain aortic homeostasis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Foundation for Cardiac Surgery (“VZW Fonds voor Hartchirurgie”), grant No. 489644Baillet-Latour Grant for Medical Research

Latent TGF-beta binding protein-1 plays an important role in craniofacial development.

Objective:This study aims to replicate the phenotype of Ltbp1 knockout mice in zebrafish, and to address the function of LTBP1 in craniofacial development.Methods:Whole mount in situ hybridization (WISH) of ltbp1 was performed at critical periods of zebrafish craniofacial development to explore the spatial-temporal expression pattern. Furthermore, we generated morpholino based knockdown model of ltbp1 to study the craniofacial phenotype.Results:WISH of ltbp1 was mainly detected in the mandibular jaw region, brain trunk, and internal organs such as pancreas and gallbladder. And ltbp1 colocalized with both sox9a and ckma in mandibular region. Morpholino based knockdown of ltbp1 results in severe jaw malformation. Alcian blue staining revealed severe deformity of Meckel's cartilage along with the absence of ceratobranchial. Three-dimension measurements of ltbp1 morphants jaws showed decrease in both mandible length and width and increase in open mouth distance. Expression of cartilage marker sox9a and muscle marker ckma was decreased in ltbp1 morphants.Conclusions:Our experiments found that ltbp1 was expressed in zebrafish mandibular jaw cartilages and the surrounding muscles. The ltbp1 knockdown zebrafish exhibited phenotypes consistent with Ltbp1 knockout mice. And loss of ltbp1 function lead to significant mandibular jaw defects and affect both jaw cartilages and surrounding muscles.

Involvement of latent TGF-beta binding protein 4 (LTBP-4) and Fibulin-5 in age-associated changes in aortic morphology and function

Involvement of latent TGF-beta binding protein 4 (LTBP-4) and Fibulin-5 in age-associated changes in aortic morphology and function

Abstract 259: Aneurysm Development in Patients With a Bicuspid Aortic Valve Is Not Associated With Transforming Growth Factor-β Activation

Objective: Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysms. Transforming growth factor-β (TGFβ) is a crucial factor of vascular remodeling, the impaired signaling of which can alter the structure and composition of the extracellular matrix. In this study, we analyzed the activity of TGFβ in aneurysmal and non-aneurysmal ascending aorta from BAV patients, using tricuspid aortic valve (TAV) patients as a reference group. Approach and Results: The response to exogenous TGFβ was analyzed with regard to gene expression in primary aortic smooth muscle cells that were isolated from 7 BAV and 5 TAV patients and in valve fibroblasts from 7 BAV and 8 TAV patients. The set of genes that were significantly changed by TGFβ (217 genes) was compared with gene expression profiles of the ascending aorta from BAV and TAV patients (139 arrays). By principle component analysis, based on the 217 genes, gene expression differed significantly in the intima/media region between aneurysmal BAV and TAV aortas, driven by the response in TAV patients. During the development of aneurysm, the levels of phosphorylated SMADs and the availability of free TGFβ were lower in BAV patients compared with TAV patients. Confocal microscopy analysis showed a higher co-localization of latency associated peptide and latent TGF-beta binding protein 3 in BAV aortas. Conclusions: Our findings suggest that TGFβ activation during aneurysm formation is muted in patients with BAV and, by interfering with repair processes, could explain the greater propensity of BAV to aortic complications.

9 Matrix remodeling in head and neck squamous cell carcinomais

Extracellular matrix (ECM) remodeling is an inherent characteristic of invasive head and neck squamous cell carcinoma (HNSCC). We investigated the regulation of gene expression, synthesis and activation of ECM components and matrix remodeling enzymes in a co-culture model of head and neck squamous cell carcinoma (HNSCC) and in HNSCC tissue. Periodontal ligament fibroblasts (PDL) and SCC-25 HNSCC cells were co-cultured for seven days, followed by mRNA and protein expression analysis of ECM components, ECM receptors, matrix metalloproteinases (MMPs) and proteinase inhibitors. The activity of MMPs was analyzed using gelatinase zymography. MMP-9, fibronectin and “antiadhesion proteins” were investigated by immunohistochemistry in HNSCC tissue. In co-cultured fibroblasts, in addition to fibronectin (FN), the “antiadhesion proteins” tenascin C (TSC), thrombospondin 1 (TSP1) and latent TGF-beta binding protein 2 (LTBP2) were upregulated. In lower levels: FN, TSC and TSP1 were also produced by the tumor cells. In co-cultured fibroblasts, alpha 1 and 5 and beta 1 integrins, in co-cultured tumor cells alpha 3 and 5 integrins were upregulated. MMP-1 was upregulated in both co-cultured tumor cells and fibroblasts, MMP-2 in co-cultured fibroblasts, and MMP-9 in co-cultured tumor cells. MMP inhibitors were mainly produced in fibroblasts. The activation of MMP-2 required the co-culture with tumor cells. MMP-9 was exclusively produced in tumor cells. In sections of formalin-fixed paraffin embedded HNSCC tissue, FN and LTBP2 were detected in fibroblastic stroma surrounding tumor cell nests; TSP1 was detected both in tumor cell nests and in the surrounding fibroblastic stroma. Extended MMP-9 immunohistochemical staining was found in 27 of 280 HNSCC tissue samples, in a subpopulation of cells in tumor cell nests. Fibroblast – tumor cell communication is a major pathway of matrix remodeling in HNSCC tumor tissue. Carcinoma-associated fibroblasts produce new matrix elements as TSP1, TSC and LTBP2, both tumor cells and fibroblasts upregulate integrin receptors, and they also share tasks in synthesis and activation of matrix remodeling enzymes. Intensive matrix re-organization takes place at the borders of tumor cell nests.

M.I.2 The matrix as a modifier for muscular dystrophy

Duchenne muscular dystrophy and the limb girdle muscular dystrophies, like other Mendelian disorders, are modified by genetic and environmental factors. We set out to identify modifiers of muscular dystrophy using a mouse model of LGMD 2C. Mice deleted for gamma sarcoglycan (Sgcg), a dystrophin associated protein, were used for mapping genetic modifiers because humans with LGMD 2C often display a variable phenotype despite having the same identical mutation. We used an intercross between Sgcg mice in the DBA2J background, which confers a severe phenotype, and Sgcg mice in the 129T2/SvEmsJ, which confers a mild background. A genomewide scan identified a locus on murine chromosome 7 that modified two different aspects of muscular dystrophy pathology. The chromosome 7 locus regulated fibrosis and also Evans blue dye uptake into skeletal muscle, a marker of sarcolemmal leakage. An insertion/deletion polymorphism was identified in the Ltbp4 gene, and this polymorphism correlated with severity of muscular dystrophy. In collaboration with the United Dystrophinopathy project, we queried whether human LTBP4 genotype correlated with outcome in DMD patients. We identified that LTBP4 acted as a modifier of DMD. LTBP4 is the latent TGFbeta binding protein and a member of the fibrillin superfamily. The members of this family reside within the extracellular matrix where they participate in binding and sequestering TGFbeta proteins. Release of TGFbeta proteins from the matrix permits ligand binding to cell surface TGFbeta receptors and activation of intracellular TGFbeta signaling. Together, these data, from mouse models and human patients, support that hyper-TGFbeta signaling is pathogenic in DMD and LGMDs, and suggest that reduction of TGFbeta signaling may be therapeutic.

Expression of Fibrillins, Latent TGFbeta Binding Proteins and TGFbetas in the Bovine Fetal Ovary.

Expression of Fibrillins, Latent TGFbeta Binding Proteins and TGFbetas in the Bovine Fetal Ovary.

Distribution of mRNAs encoding transforming growth factors‐β1, ‐2, and ‐3 in the intact rat brain and after experimentally induced focal ischemia

Transforming growth factors-beta1 (TGF-beta1), -2, and -3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF-betas were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF-betas, and in situ hybridization was performed. We demonstrated high TGF-beta1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF-beta2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF-beta3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF-betas with markedly different expression patterns. TGF-beta1 was induced in the penumbral region of cortex and striatum, whereas TGF-beta2 and -beta3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF-betas in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF-beta binding proteins. Furthermore, they might have subtype-specific functions following ischemic attack.

Low‐dose ultraviolet radiation selectively degrades chromophore‐rich extracellular matrix components

Photoageing of human skin due to chronic exposure to ultraviolet radiation (UVR) is characterized histologically by extensive remodelling of the dermal elastic fibre system. Whilst enzymatic pathways are thought to play a major role in mediating extracellular matrix (ECM) degeneration in UV-exposed skin, the substrate specificity of UVR-up-regulated and activated matrix metalloproteinases (MMPs) is low. It is unclear, therefore, how such cell-mediated mechanisms alone could be responsible for the reported selective degradation of elastic fibre components such as fibrillin-1 and fibulin-5 during the early stages of photoageing. Here we use atomic force microscopy (AFM) and scanning transmission electron microscopy (STEM) to demonstrate that physiologically attainable doses (20-100 mJ/cm(2)) of direct UV-B radiation can induce profound, dose-dependent, changes in the structure of, and mass distribution within, isolated fibrillin microfibrils. Furthermore, using reducing and native PAGE in combination with AFM, we show that, whilst exposure to low-dose UV-B radiation significantly alters the macromolecular and quaternary structures of both UV chromophore (Cys, His, Phe, Trp and Tyr)-rich fibrillin microfibrils (fibrillin-1, 21.0%) and fibronectin dimers (fibronectin, 12.9%), similar doses have no detectable effect on UV chromophore-poor type I collagen monomers (2.2%). Analysis of the published primary amino acid sequences of 49 dermal ECM components demonstrates that most elastic fibre-associated proteins, but crucially neither elastin nor members of the collagen family, are rich in UV chromophores. We suggest, therefore, that the amino acid composition of elastic fibre-associated proteins [including the fibrillins, fibulins, latent TGFbeta binding proteins (LTBPs) and the lysyl oxidase family of enzymes (LOK/LOXLs)] may predispose them to direct degradation by UVR. As a consequence, this selective acellular photochemical pathway may play an important role in initiating and/or exacerbating cell-mediated ECM remodelling in UVR-exposed skin.

LTBP2 null mutations in an autosomal recessive ocular syndrome with megalocornea, spherophakia, and secondary glaucoma

The latent TGFβ-binding proteins (LTBPs) and fibrillins are a superfamily of large, multidomain proteins with structural and TGFβ-signalling roles in the extracellular matrix. Their importance is underscored by fibrillin-1 mutations responsible for Marfan syndrome, but their respective roles are still incompletely understood. We report here on two families where children from healthy, consanguineous parents, presented with megalocornea and impaired vision associated with small, round, dislocated lenses (microspherophakia and ectopia lentis) and myopia, as well as a high-arched palate, and, in older children, tall stature with an abnormally large arm span over body height ratio, that is, associated features of Marfan syndrome. Glaucoma was not present at birth, but was diagnosed in older children. Whole genome homozygosity mapping followed by candidate gene analysis identified homozygous truncating mutations of LTBP2 gene in patients from both families. Fibroblast mRNA analysis was consistent with nonsense-mediated mRNA decay, with no evidence of mutated exon skipping. We conclude that biallelic null LTBP2 mutations cause the ocular phenotype in both families and could lead to Marfan-like features in older children. We suggest that intraocular pressures should be followed-up in young children with an ocular phenotype consisting of megalocornea, spherophakia and/or lens dislocation, and recommend LTBP2 gene analysis in these patients.

Independent regulation of short and long forms of latent TGF‐β binding protein (LTBP)‐4 in cultured fibroblasts and human tissues

Transforming growth factor (TGF)-beta is secreted and targeted into the extracellular matrix (ECM) in association with one of the latent TGF-beta binding proteins (LTBPs). Activation of these latent complexes is an important regulatory step in TGF-beta signaling. LTBPs target the growth factor into the ECM and expose it to activating mechanisms. Disruption of LTBP-4 gene causes severe developmental abnormalities in both humans and mice. Transcripts for two N-terminally distinct LTBP-4 variants, LTBP-4S (short) and -4L (long), have been identified. In the current work, we have characterized differences in the expression, processing, and ECM targeting of these LTBP-4 variants. Heart and skeletal muscle displayed expression of both variants, while liver expressed mainly LTBP-4L and lung as well as small intestine LTBP-4S. This tissue-specific expression pattern was found to originate from control of transcription by two independent promoters. Furthermore, LTBP-4S and -4L proteins were secreted and processed differently. During secretion, LTBP-4L was complexed with TGF-beta1, whereas the majority of LTBP-4S was secreted in a free form. In addition, LTBP-4S was incorporated into the ECM, while full-length LTBP-4L was not readily detectable in the ECM. These data suggest that LTBP-4 functions are modified by tissue-specific expression of the two N-terminally distinct variants, which in addition exhibit significant differences in cellular processing and targeting, that is, this provides a basis for understanding molecular diversity in LTBP-4 structure and function.

Alteration of Function for Transforming Growth Factor (TGF)-Beta1 by Latent TGF-Beta Binding Protein Yields Inframatory Bowel Disease and Tumors

Alteration of Function for Transforming Growth Factor (TGF)-Beta1 by Latent TGF-Beta Binding Protein Yields Inframatory Bowel Disease and Tumors

Matrix association of latent TGF‐beta binding protein‐2 (LTBP‐2) is dependent on fibrillin‐1

The components of the extracellular matrix (ECM) and their differential expression patterns play important roles in tissue formation. The deposition of latent TGF-beta binding proteins (LTBPs) to the ECM exhibit distinct distribution profiles. We have analyzed here the temporal and spatial ECM association of latent TGF-beta binding protein LTBP-2 in cultured human embryonic lung fibroblasts. We found that LTBP-2 was not assembled to the ECM until by confluency of cultures following the deposition of fibronectin (FN) and fibrillin-1. In 5-day-old cultures LTBP-2 was rapidly secreted from cells and it subsequently associated with the ECM as shown by metabolic labeling and immunoprecipitation. LTBP-2 colocalized transiently with fibronectin and failed to assemble to the ECM of FN deficient mouse fibroblasts. Analysis of different cultured human cell lines revealed partial colocalization of LTBP-2 and fibrillin-1 in the ECM of fibroblasts, MG-63 osteosarcoma cells and human vascular endothelial cells. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2. Current results suggest that LTBP-2 is not an element of the provisional ECM of fibroblasts but is more likely a component of more mature ECM and indicate that matrix association of LTBP-2 depends on a pre-formed fibrillin-1 network.

Modulation of TGF‐β activity by latent TGF‐β‐binding protein 1 in human malignant glioma cells

High biological activity of the transforming growth factor (TGF)-beta-Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF-beta has become a novel target for the experimental treatment of these tumors. TGF-beta is processed by furin-like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency-associated peptide (LAP). Latent TGF-beta-binding protein 1 (LTBP-1) covalently binds to this small latent TGF-beta complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP-1 protein in vivo increase with the grade of malignancy in gliomas. LTBP-1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP-1 into the medium is decreased by the inhibition of FLP activity. Gene-transfer mediated overexpression of LTBP-1 in glioma cell lines results in an increase inTGF-beta activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF-beta activity is enhanced. Conversely, LTBP-1 gene silencing reduces TGF-beta activity and Smad2 phosphorylation without affecting TGF-beta protein levels. Collectively, we identify LTBP-1 as an important modulator of TGF-beta activation in glioma cells, which may contribute to the malignant phenotype of these tumors.

Oligodontia Is Caused by Mutation in LTBP3, the Gene Encoding Latent TGF-β Binding Protein 3

We have identified a consanguineous Pakistani family where oligodontia is inherited along with short stature in an autosomal-recessive fashion. Increased bone density was present in the spine and at the base of the skull. Using high-density single-nucleotide polymorphism microarrays for homozygosity mapping, we identified a 28 Mb homozygous stretch shared between affected individuals on chromosome 11q13. Screening selected candidate genes within this region, we identified a homozygous nonsense mutation, Y774X, within LTBP3, the gene for the latent TGF-beta binding protein 3, an extracellular matrix protein believed to be required for osteoclast function.

Marfan syndrome and its disorder in periodontal tissues

Elastic system fibers are composed of two distinct elements, elastin, which is an amorphous component crosslinked in the core, and microfibril, localized in the periphery of elastin. As microfibrillar proteins, fibrillins, microfibril-associated glycoproteins (MAGPs), latent TGF-beta-binding proteins (LTBPs), microfibril-associated proteins (MFAPs), and fibulins are known. Fibrillin-1 is a major microfibrillar protein and characterized by calcium binding EGF-like (cbEGF) domain. Association between fibrillin-1 and TGF-beta is a recent topic of this field and this interaction is known to inactivate and target TGF-beta action. FBN1 encoding fibrillin-1 is a responsible gene for Marfan syndrome type 1 (MIM #154700), characterized by increased height and long limbs, ectopia lentis, and cardiovascular disorders, such as mitral valve prolapse and aortic dilation and regurgitation. Animal models suggest that the abnormal TGF-beta signaling is underlying as the pathogenesis of these conditions. Besides skeletal, ocular and cardiovascular conditions, severe periodontitis is frequently seen in affected patients. To clarify the unknown function of elastic system fibers in the periodontal ligament (PDL), PDL-cells were isolated from a Marfan syndrome type 1 patient who was with the severe periodontitis and had a mutation in one of the cbEGF domain of fibrillin-1. These results suggested that wild-type fibrillin-1 was required for the normal cell alignment and tissue architecture of PDLs. Evidences are now accumulated to suggest that fibrillin-1 is one of the molecule involved in the interaction between cell and extracellular matrix.

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis.

Perturbation of transforming growth factor (TGF)-beta1 association with latent TGF-beta binding protein yields inflammation and tumors.

Transforming growth factor-beta (TGF-beta) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-beta is often released as part of an inactive tripartite complex consisting of TGF-beta, the TGF-beta propeptide, and a molecule of latent TGF-beta binding protein (LTBP). The interaction of TGF-beta and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-beta from this state is crucial for signaling. To examine the contribution of LTBP to TGF-beta function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1(C33S/C33S) mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-beta1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1(-/-) animals. Tgfb1(C33S/C33S) mice exhibited decreased levels of active TGF-beta1, decreased TGF-beta signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-beta complex is important for proper TGF-beta1 function and that Tgfb1(C33S/C33S) mice are hypomorphs for active TGF-beta1. Moreover, although mechanisms exist to activate latent TGF-beta1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.

Dual functions for LTBP in lung development: LTBP‐4 independently modulates elastogenesis and TGF‐β activity

The latent TGF-beta binding proteins (LTBP) -1, -3, and -4 are extracellular proteins that assist in the secretion and localization of latent TGF-beta. The null mutation of LTBP-4S in mice causes defects in the differentiation of terminal air-sacs, fragmented elastin, and colon carcinomas. We investigated lung development from embryonic day 14.5 (E14.5) to day 7 after birth (P7) in order to determine when the defects in elastin organization initiate and to further examine the relation of TGF-beta signaling levels and air-sac septation in Ltbp4S-/- lungs. We found that defects in elastogenesis are visible as early as E14.5 and are maintained in the alveolar walls, in blood vessel media, and subjacent airway epithelium. The air-sac septation defect was associated with excessive TGF-beta signaling and was reversed by lowering TGF-beta2 levels. Thus, the phenotype is not directly reflective of a change in TGF-beta1, the only TGF-beta isoform known to complex with LTBP-4. Reversal of the air-sac septation defect was not associated with normalization of the elastogenesis indicating two separate functions of LTBP-4 as a regulator of elastic fiber assembly and TGF-beta levels in lungs.

Mice Lacking Neutrophil Elastase Are Resistant to Bleomycin-Induced Pulmonary Fibrosis

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.