ABSTRACT
Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFß signaling. TGFß is secreted from cells as a latent complex consisting of TGFß, the TGFß propeptide, and a molecule of latent TGFß binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFß levels, and has been hypothesized as an explanation for enhanced TGFß signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFß signaling in MFS might be due to defective interaction of latent TGFß complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFß complexes composed of LTBP-3 and TGFß contributes to aortic disease progression.
Subject(s)
Aortic Aneurysm, Thoracic/metabolism , Latent TGF-beta Binding Proteins/metabolism , Marfan Syndrome/complications , Marfan Syndrome/genetics , Multiprotein Complexes/metabolism , Transforming Growth Factor beta/metabolism , Analysis of Variance , Animals , Aortic Aneurysm, Thoracic/etiology , DNA, Complementary/biosynthesis , Fibrillin-1 , Fibrillins , Immunohistochemistry , Latent TGF-beta Binding Proteins/deficiency , Mice , Microfilament Proteins/genetics , Muscle, Smooth, Vascular/cytology , Real-Time Polymerase Chain ReactionABSTRACT
Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFß) signaling. Despite elevated extracellular TGFß activity, downstream signaling molecules of the TGFß pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFß receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFß1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFß receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFß signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFß receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFß receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.
Subject(s)
Cutis Laxa/genetics , Latent TGF-beta Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Animals , Case-Control Studies , Cells, Cultured , Disease Models, Animal , Down-Regulation , Endocytosis/genetics , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Immunoprecipitation , Latent TGF-beta Binding Proteins/genetics , Male , Mice , Mice, Knockout , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Receptor, Transforming Growth Factor-beta Type I , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/genetics , Signal Transduction , Smad2 Protein/genetics , Smad2 Protein/metabolismABSTRACT
Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.
Subject(s)
Amelogenesis Imperfecta/genetics , Latent TGF-beta Binding Proteins/genetics , Osteochondrodysplasias/genetics , Adolescent , Amelogenesis Imperfecta/diagnostic imaging , Animals , Base Sequence , Child , Consanguinity , DNA Mutational Analysis , Female , Frameshift Mutation , Genetic Association Studies , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation, Missense , Osteochondrodysplasias/diagnostic imaging , Pedigree , Radiography , Sequence DeletionABSTRACT
Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-ß (TGF-ß). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-ß signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.
Subject(s)
Amelogenesis/genetics , Dental Enamel/abnormalities , Latent TGF-beta Binding Proteins/genetics , Ameloblasts/metabolism , Amelogenesis Imperfecta/genetics , Animals , Dental Enamel/ultrastructure , Genotype , Male , Mice , Mice, Mutant Strains , Microscopy, Electron, Scanning , Mutation , Osteochondrodysplasias/genetics , Phenotype , Tooth Calcification/genetics , Transforming Growth Factor beta/genetics , X-Ray MicrotomographyABSTRACT
Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-ß binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-ß because TGF-ß-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-ß-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.
Subject(s)
Extracellular Matrix Proteins/metabolism , Latent TGF-beta Binding Proteins/physiology , Recombinant Proteins/metabolism , Animals , HEK293 Cells , Humans , Latent TGF-beta Binding Proteins/metabolism , Mice , Mice, Knockout , Protein Binding , RNA InterferenceABSTRACT
Mice deficient in Latent TGFß Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFß2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFß1 or TGFß3 did not improve lung septation indicating that the TGFß isoform elevated in Ltbp4S(-/-) lungs is TGFß2. Expression of a form of Ltbp4 that could not bind latent TGFß did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFß complexes. Therefore, the change in TGFß-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGFß2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly.
Subject(s)
Body Patterning/genetics , Elastic Tissue/embryology , Extracellular Matrix Proteins/physiology , Latent TGF-beta Binding Proteins/physiology , Lung/embryology , Transforming Growth Factor beta2/metabolism , Animals , Elastic Tissue/abnormalities , Elastin/metabolism , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Fibrillins , Latent TGF-beta Binding Proteins/genetics , Lung/abnormalities , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/metabolism , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Recombinant Proteins/genetics , Signal Transduction/genetics , Transforming Growth Factor beta2/geneticsABSTRACT
Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.
Subject(s)
Cancer Pain , Mouth Neoplasms , Peptide Hormones , Humans , Mice , Animals , Calcitonin Gene-Related Peptide/metabolism , Calcitonin , Procalcitonin , Receptors, Calcitonin Gene-Related Peptide/genetics , Receptors, Calcitonin Gene-Related Peptide/metabolism , Cancer Pain/drug therapy , Endothelial Cells/metabolism , Mouth Neoplasms/drug therapy , Tumor MicroenvironmentABSTRACT
Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin-1 and fibrillin-2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril-associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF-ß binding protein 1 (LTBP-1), -3 and -4; the three LTBPs that form a complex with TGF-ß. In Fbn1(-/-) ascending aortas and lungs, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. In addition, in cultures of Fbn1(-/-) smooth muscle cells or lung fibroblasts, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. Fibrillin-2 is not involved in the deposition of LTBP-1 in Fbn1(-/-) extracellular matrix as cells deficient for both fibrillin-1 and fibrillin-2 still incorporate LTBP-1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1(-/-) cells abrogates the deposition of LTBP-1. Together, these data indicate that LTBP-3 and LTBP-4 association with the matrix depends on fibrillin-1 microfibrils, whereas LTBP-1 association depends on a fibronectin network.
Subject(s)
Fibronectins/metabolism , Gene Expression Regulation/physiology , Latent TGF-beta Binding Proteins/metabolism , Microfilament Proteins/metabolism , Animals , DNA, Complementary/genetics , DNA, Complementary/metabolism , Fibrillin-1 , Fibrillin-2 , Fibrillins , Fibroblasts/metabolism , Fibronectins/genetics , Latent TGF-beta Binding Proteins/genetics , Lung/cytology , Mice , Mice, Knockout , Microfilament Proteins/genetics , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , RNA/genetics , RNA/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.
Subject(s)
Dermis/abnormalities , Intestines/abnormalities , Latent TGF-beta Binding Proteins/genetics , Lung/abnormalities , Mutation , Urinary Tract/abnormalities , Cells, Cultured , Child , Child, Preschool , Coculture Techniques , Culture Media, Conditioned/chemistry , DNA/genetics , DNA/isolation & purification , Dermis/metabolism , Dermis/ultrastructure , Female , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Heterozygote , Homozygote , Humans , Immunohistochemistry , Infant , Intestinal Mucosa/metabolism , Latent TGF-beta Binding Proteins/chemistry , Lung/metabolism , Male , Musculoskeletal System , Protein Structure, Tertiary , RNA, Messenger/metabolism , Sequence Analysis, DNA , Skin/cytology , Syndrome , Urinary Tract/metabolismABSTRACT
The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alpha v beta6 and alpha v beta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system.
Subject(s)
Integrins/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Binding Sites , Mice , Mice, Knockout , Phenotype , Transforming Growth Factor beta1/chemistry , Transforming Growth Factor beta1/genetics , Vascular Diseases/genetics , Vascular Diseases/pathology , Yolk Sac/blood supply , Yolk Sac/pathologyABSTRACT
Transforming growth factor-beta (TGFß) is released from cells as part of a trimeric latent complex consisting of TGFß, the TGFß propeptides, and either a latent TGFß binding protein (LTBP) or glycoprotein-A repetitions predominant (GARP) protein. LTBP1 and 3 modulate latent TGFß function with respect to secretion, matrix localization, and activation and, therefore, are vital for the proper function of the cytokine in a number of tissues. TGFß modulates stem cell differentiation into adipocytes (adipogenesis), but the potential role of LTBPs in this process has not been studied. We observed that 72â¯h post adipogenesis initiation Ltbp1, 2, and 4 expression levels decrease by 74-84%, whereas Ltbp3 expression levels remain constant during adipogenesis. We found that LTBP3 silencing in C3H/10T1/2 cells reduced adipogenesis, as measured by the percentage of cells with lipid vesicles and the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Lentiviral mediated expression of an Ltbp3 mRNA resistant to siRNA targeting rescued the phenotype, validating siRNA specificity. Knockdown (KD) of Ltbp3 expression in 3T3-L1, M2, and primary bone marrow stromal cells (BMSC) indicated a similar requirement for Ltbp3. Epididymal and inguinal white adipose tissue fat pad weights of Ltbp3-/- mice were reduced by 62% and 57%, respectively, compared to wild-type mice. Inhibition of adipogenic differentiation upon LTBP3 loss is mediated by TGFß, as TGFß neutralizing antibody and TGFß receptor I kinase blockade rescue the LTBP3 KD phenotype. These results indicate that LTBP3 has a TGFß-dependent function in adipogenesis both in vitro and possibly in vivo. SIGNIFICANCE: Understanding the control of mesenchymal stem cell fate is crucial for the potential use of these cells for regenerative medicine.
Subject(s)
Adipogenesis , PPAR gamma , Adipogenesis/genetics , Animals , Antibodies, Neutralizing , Cell Differentiation , Lipids , Mice , Mice, Inbred C3H , PPAR gamma/genetics , PPAR gamma/metabolism , RNA, Messenger/genetics , RNA, Small Interfering , Transcription Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta3 , Transforming Growth FactorsABSTRACT
The latent TGF-ß binding proteins (LTBP-1 -3, and -4) assist in the secretion and localization of latent TGF-ß molecules. Ltbp3(-/-) and Ltbp4S(-/-) mice have distinct phenotypes and only in the lungs does deficiency of either Ltbp-3 or Ltbp-4 cause developmental abnormalities. To determine if these two LTBPs have additional common functions, we generated mice deficient for both Ltbp-3 and Ltbp-4S. The only novel defect in Ltbp3(-/-);Ltbp4S(-/-) mice was an early lethality compared to mice with single mutations. In addition lung abnormalities were exacerbated and the terminal air sac septation defect was more severe in Ltbp3(-/-);Ltbp4S(-/-) mice than in Ltbp4S(-/-) mice. Decreased cellularity of Ltbp3(-/-);Ltbp4S(-/-) lungs was correlated with higher rate of apoptosis in newborn lungs of Ltbp3(-/-);Ltbp4S(-/-) animals compared to WT, Ltbp3(-/-), and Ltbp4S(-/-) mice. No differences in the maturation of the major lung cell types were discerned between the single and double mutant mice. However, the distribution of type 2 cells and myofibroblasts was abnormal, and myofibroblast segregation in some areas might be an indication of early fibrosis. We also observed differences in ECM composition between Ltbp3(-/-);Ltbp4S(-/-) and Ltbp4S(-/-) lungs after birth, reflected in decreased incorporation of fibrillin-1 and -2 in Ltbp3(-/-);Ltbp4S(-/-) matrix. The function of the lungs of Ltbp3(-/-);Ltbp4S(-/-) mice after the first week of life was potentially further compromised by macrophage infiltration, as proteases secreted from macrophages might exacerbate developmental emphysema. Together these data indicate that LTBP-3 and -4 perform partially overlapping functions only in the lungs.
Subject(s)
Latent TGF-beta Binding Proteins/metabolism , Lung/embryology , Lung/metabolism , Animals , Apoptosis , Cell Differentiation , Elastin/biosynthesis , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Inflammation/metabolism , Inflammation/pathology , Latent TGF-beta Binding Proteins/deficiency , Latent TGF-beta Binding Proteins/genetics , Lung/pathology , Mice , Microfibrils/metabolism , PhenotypeABSTRACT
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.
Subject(s)
Inflammation/metabolism , Latent TGF-beta Binding Proteins/metabolism , Neoplasms/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Cells, Cultured , Fibroblasts/cytology , Gastric Mucosa/cytology , Gastric Mucosa/metabolism , Gastric Mucosa/pathology , Inflammation/pathology , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Langerhans Cells/cytology , Langerhans Cells/metabolism , Latent TGF-beta Binding Proteins/genetics , Mice , Mice, Knockout , Neoplasms/pathology , Protein Precursors/genetics , Protein Precursors/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta1/geneticsABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14 -/- ) and man causes dwarfism, osteopenia, arthritis, and lipodystrophy, abnormalities ascribed to defective collagen turnover. We have previously shown non-proteolytic functions of MT1-MMP mediated by its cytoplasmic tail, where the unique tyrosine (Y573) controls intracellular signaling. The Y573D mutation blocks TIMP-2/MT1-MMP-induced Erk1/2 and Akt signaling without affecting proteolytic activity. Here, we report that a mouse with the MT1-MMP Y573D mutation (Mmp14 Y573D/Y573D ) shows abnormalities similar to but also different from those of Mmp14 -/- mice. Skeletal stem cells (SSC) of Mmp14 Y573D/Y573D mice show defective differentiation consistent with the mouse phenotype, which is rescued by wild-type SSC transplant. These results provide the first in vivo demonstration that MT1-MMP modulates bone, cartilage, and fat homeostasis by controlling SSC differentiation through a mechanism independent of proteolysis.
ABSTRACT
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.
Subject(s)
Elastin/metabolism , Latent TGF-beta Binding Proteins/metabolism , Lung/embryology , Lung/growth & development , Protein Isoforms/metabolism , Transforming Growth Factor beta/metabolism , Animals , Benzamides/metabolism , Dioxoles/metabolism , Elastin/genetics , Elastin/ultrastructure , Female , Latent TGF-beta Binding Proteins/genetics , Lung/anatomy & histology , Lung/metabolism , Male , Mice , Mice, Knockout , Phenotype , Pregnancy , Protein Isoforms/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pulmonary Alveoli/physiology , Pulmonary Alveoli/ultrastructure , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/geneticsABSTRACT
The TGF-betas are multifunctional proteins whose activities are believed to be controlled by interaction with the latent TGF-beta binding proteins (LTBPs). In spite of substantial effort, the precise in vivo significance of this interaction remains unknown. To examine the role of the Ltbp-3, we made an Ltbp-3-null mutation in the mouse by gene targeting. Homozygous mutant animals develop cranio-facial malformations by day 10. At 2 mo, there is a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. Histological examination of the skulls from null animals revealed ossification of the synchondroses within 2 wk of birth, in contrast to the wild-type synchondroses, which never ossify. Between 6 and 9 mo of age, mutant animals also develop osteosclerosis and osteoarthritis. The pathological changes of the Ltbp-3-null mice are consistent with perturbed TGF-beta signaling in the skull and long bones. These observations give support to the notion that LTBP-3 is important for the control of TGF-beta action. Moreover, the results provide the first in vivo indication for a role of LTBP in modulating TGF-beta bioavailability.
Subject(s)
Adaptor Proteins, Signal Transducing , Bone and Bones/abnormalities , Bone and Bones/metabolism , Carrier Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Bone Remodeling , Bone and Bones/pathology , Carrier Proteins/genetics , Craniofacial Abnormalities/complications , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/pathology , Gene Deletion , Gene Targeting , In Situ Hybridization , Latent TGF-beta Binding Proteins , Mice , Mice, Knockout , Osteoarthritis/complications , Osteoarthritis/genetics , Osteoarthritis/pathology , Osteosclerosis/complications , Osteosclerosis/genetics , Osteosclerosis/pathology , RNA, Messenger/analysis , RNA, Messenger/genetics , Skull/abnormalities , Skull/metabolism , Skull/pathologyABSTRACT
Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFß, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.
Subject(s)
Latent TGF-beta Binding Proteins/physiology , Neoplasms/genetics , Neoplasms/pathology , Animals , Cell Line, Tumor , Chick Embryo , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Latent TGF-beta Binding Proteins/antagonists & inhibitors , Latent TGF-beta Binding Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasms/mortality , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , RNA, Small Interfering/pharmacology , Survival AnalysisABSTRACT
Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs' cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-ß) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-ß signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-ß signaling pathway, was found to be miR21-5p's putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-ß signaling pathway and stimulation of expression of miR21-5p in hBMSCs.
ABSTRACT
The transforming growth factor (TGF)-betas are essential in pre- and postnatal development, differentiation and morphogenesis of higher organisms. Quantitation of the levels of TGF-beta synthesis, secretion, and activation are crucial for grasping the mechanisms that control these events and ultimately control TGF-beta action. Rather than presenting a single method, we describe several methods for measuring active TGF-beta in different experimental situations. This is possible as a result of advances in transgenic mice technology that allow in vivo TGF-beta measurements in addition to the more established in vitro approaches.
Subject(s)
Genetic Techniques , Molecular Biology/methods , Transforming Growth Factor beta/analysis , Transforming Growth Factor beta/biosynthesis , Animals , Coculture Techniques , DNA-Binding Proteins/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/cytology , Green Fluorescent Proteins/metabolism , Immunohistochemistry , Luciferases/metabolism , Lung/cytology , Mice , Mice, Transgenic , Mink , Smad Proteins , Trans-Activators/metabolism , Transforming Growth Factor beta/geneticsABSTRACT
The LTBPs (or latent transforming growth factor ß binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFß by covalently binding the TGFß propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFß precursor in the trans-golgi network but LAP and TGFß remain strongly bound through non-covalent interactions. LAP, TGFß, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFß latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFß activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFß regulation, TGFß-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.