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Long form of latent TGF-beta binding protein 1 (Ltbp1L) regulates cardiac valve development

Todorovic, Vesna; Finnegan, Erin; Freyer, Laina; Zilberberg, Lior; Ota, Mitsuhiko; Rifkin, Daniel B
Transforming Growth Factor beta (TGF-beta) is crucial for valve development and homeostasis. The long form of Latent TGF-beta binding protein 1 (LTBP1L) covalently binds all TGF-beta isoforms and regulates their bioavailability. Ltbp1L expression analysis during valvulogenesis revealed two patterns of Ltbp1L production: an early one (E9.5-11.5) associated with endothelial-to-mesenchymal transformation (EMT); and a late one (E12.5 to birth) contemporaneous with valve remodeling. Similarly, histological analysis of Ltbp1L(-/-) developing valves identified two different pathologies: generation of hypoplastic endocardial cushions in early valvulogenesis, followed by development of hyperplastic valves in late valvulogenesis. Ltbp1L promotes valve EMT, as Ltbp1L absence yields hypoplastic endocardial cushions in vivo and attenuated EMT in vitro. Ltbp1L(-/-) valve hyperplasia in late valvuogenesis represents a consequence of prolonged EMT. We demonstrate that Ltbp1L is a major regulator of Tgf-beta activity during valvulogenesis since its absence results in a perturbed Tgf-beta pathway that causes all Ltbp1L(-/-) valvular defects. Developmental Dynamics, 2011. (c) 2010 Wiley-Liss, Inc
PMCID:3012267
PMID: 21181942
ISSN: 1097-0177
CID: 116226

Development of a novel multiplexed-TGFbeta assay

Hardee M.E.; Marciscano A.E.; Rifkin D.B.; Barcellos-Hoff M.
Purpose/Objective(s): Transforming growth factor-beta (TGFbeta) levels are increased by radiation therapy. Pre-and post-radiotherapy systemic levels of TGFb measured by ELISA, mink lung epithelial cell luciferase assay (MLEC), and cell-based biological assays have been correlated with treatment outcomes and radiation-induced toxicity. However, aspects of these assays and TGFbeta biology can make TGFb measurements difficult to interpret. TGFbeta is secreted along with the latency associated peptide (LAP) that is activated extracellularly when needed; available assays measure active TGFbeta using artificial activation by acid or heat. Specimen contamination by platelets is another source of variability since latent TGFbeta is present in platelets in large quantities. Our goal is to develop an assay that eliminates artificial activation, includes an internal control for platelet contamination, and can reproducibly measure both active and latent TGFbeta. Materials/Methods: To measure active TGFbeta1, a commercially available TGFbeta1 ELISA using the Meso Scale Discovery (MSD) platform was used. MSD uses electrochemiluminescent detection that provides sensitivity over a concentration range of 5 logs and allows multiplexing of up to 10 assays. An ELISA for LAP-b1, a measure of latent TGFbeta1 was developed. Different preparations of plasma and serum were tested and contamination from platelet degranulation was measured using an ELISA of platelet factor-4 (PF4), which is released from a-granules of activated platelets. Results: LAP-s1 and PF4 assays using the MSD platform were successfully developed using commercially available antibodies. LAP-b1 ELISA was a highly reproducible measurement of total TGFbeta, as verified using MLEC assay of exogenously activated samples. Measurement of total TGFb in serum (19,085-156,058 pg/mL) was high and variable due to contamination by platelet activation as measured by PF4. By spiking plasma with increasing volumes of serum (to replicate platelet contamination), we determined a PF4 cutoff value of 1.5 mg/mL, below which total TGFbeta levels were constant. Collection of plasma in EDTA containing tubes (vs heparin tubes) and double centrifugation was most effective at reducing platelet contamination and lead to the most reproducible TGFbeta measurements that averaged 344 pg/ml.We determined that plasma contains undetectable levels of active TGFbeta but that as little as 62 pg/mL exogenous TGFbeta could be detected in plasma. Conclusions:We developed an improved TGFbeta assay for biological fluids that simultaneously measures active and latent TGFbeta. By avoiding the uncertainty introduced by exogenous activation in currently available assays and including an internal control for platelet contamination, this assay will be more robust for future correlative studies of systemic TGFbeta levels and radiation treatment outcomes and toxicity
EMBASE:70649935
ISSN: 0360-3016
CID: 150889

F-Spondin (spondin-1) Null Mice Exhibit Increased Bone Formation. [Meeting Abstract]

Attur, Mukundan; Palmer, Glyn; Liu, James; Qing, Yang; Rifkin, Daniel; Beier, Frank; Abramson, Steven B
ISI:000297621503005
ISSN: 0004-3591
CID: 2331142

F-Spondin Mediates Catabolic Effects on Articular Chondrocytes Via Its Thrombospondin Repeat Domain. [Meeting Abstract]

Liu, James; Palmer, Glyn; Qing, Yang; Rifkin, Daniel; Attur, Mukundan; Abramson, Steven B
ISI:000297621502210
ISSN: 0004-3591
CID: 2331122

Differential effects of alendronate and losartan therapy on osteopenia and aortic aneurysm in mice with severe Marfan syndrome

Nistala, Harikiran; Lee-Arteaga, Sui; Carta, Luca; Cook, Jason R; Smaldone, Silvia; Siciliano, Gabriella; Rifkin, Aaron N; Dietz, Harry C; Rifkin, Daniel B; Ramirez, Francesco
Reduced bone mineral density (osteopenia) is a poorly characterized manifestation of pediatric and adult patients afflicted with Marfan syndrome (MFS), a multisystem disorder caused by structural or quantitative defects in fibrillin-1 that perturb tissue integrity and TGFbeta bioavailability. Here we report that mice with progressively severe MFS (Fbn1(mgR/mgR) mice) develop osteopenia associated with normal osteoblast differentiation and bone formation. In vivo and ex vivo experiments, respectively, revealed that adult Fbn1(mgR/mgR) mice respond more strongly to locally induced osteolysis and that Fbn1(mgR/mgR) osteoblasts stimulate pre-osteoclast differentiation more than wild-type cells. Greater osteoclastogenic potential of mutant osteoblasts was largely attributed to Rankl up-regulation secondary to improper TGFbeta activation and signaling. Losartan treatment, which lowers TGFbeta signaling and restores aortic wall integrity in mice with mild MFS, did not mitigate bone loss in Fbn1(mgR/mgR) mice even though it ameliorated vascular disease. Conversely, alendronate treatment, which restricts osteoclast activity, improved bone quality but not aneurysm progression in Fbn1(mgR/mgR) mice. Taken together, our findings shed new light on the pathogenesis of osteopenia in MFS, in addition to arguing for a multifaceted treatment strategy in this congenital disorder of the connective tissue
PMCID:2989889
PMID: 20871099
ISSN: 1460-2083
CID: 140040

Bone matrix to growth factors: location, location, location [Comment]

Rifkin, Daniel B; Todorovic, Vesna
The demonstration that fibrillin-1 mutations perturb transforming growth factor (TGF)-beta bioavailability/signaling in Marfan syndrome (MFS) changed the view of the extracellular matrix as a passive structural support to a dynamic modulator of cell behavior. In this issue, Nistala et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003089) advance this concept by demonstrating how fibrillin-1 and -2 regulate TGF-beta and bone morphogenetic protein (BMP) action during osteoblast maturation
PMCID:3101590
PMID: 20855500
ISSN: 1540-8140
CID: 112559

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta

Yang, Tao; Mendoza-Londono, Roberto; Lu, Huifang; Tao, Jianning; Li, Kaiyi; Keller, Bettina; Jiang, Ming Ming; Shah, Rina; Chen, Yuqing; Bertin, Terry K; Engin, Feyza; Dabovic, Branka; Rifkin, Daniel B; Hicks, John; Jamrich, Milan; Beaudet, Arthur L; Lee, Brendan
The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis
PMCID:2898604
PMID: 20530870
ISSN: 1558-8238
CID: 146002

Microfibril structure masks fibrillin-2 in postnatal tissues

Charbonneau, Noe L; Jordan, C Diana; Keene, Douglas R; Lee-Arteaga, Sui; Dietz, Harry C; Rifkin, Daniel B; Ramirez, Francesco; Sakai, Lynn Y
Fibrillin microfibrils are polymeric structures present in connective tissues. The importance of fibrillin microfibrils to connective tissue function has been demonstrated by the multiple genetic disorders caused by mutations in fibrillins and in microfibril-associated molecules. However, knowledge of microfibril structure is limited, largely due to their insolubility. Most previous studies have focused on how fibrillin-1 is organized within microfibril polymers. In this study, an immunochemical approach was used to circumvent the insolubility of microfibrils to determine the role of fibrillin-2 in postnatal microfibril structure. Results obtained from studies of wild type and fibrillin-1 null tissues, using monoclonal and polyclonal antibodies with defined epitopes, demonstrated that N-terminal fibrillin-2 epitopes are masked in postnatal microfibrils and can be revealed by enzymatic digestion or by genetic ablation of Fbn1. From these studies, we conclude that fetal fibrillin polymers form an inner core within postnatal microfibrils and that microfibril structure evolves as growth and development proceed into the postnatal period. Furthermore, documentation of a novel cryptic site present in EGF4 in fibrillin-1 underscores the molecular complexity and tissue-specific differences in microfibril structure.
PMCID:2888437
PMID: 20404337
ISSN: 0021-9258
CID: 163510

Binding of apoptotic fetal cardiocytes by anti-Ro/La antibodies stimulates uPA/uPAR-dependent activation of TGFbeta and potentiates fibrosis [Meeting Abstract]

Briassouli P.; Rifkin D.; Buyon J.P.; Clancy R.M.
Purpose: Organ injury induced by antibodies characteristic of Sjogren Syndrome and Systemic Lupus Erythematosus, while varied in the adult and fetus, may share in common a link between apoptosis and ultimate fibrosis. In congenital heart block (CHB), binding of maternal anti-Ro/La antibodies to apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Immunological staining of CHB hearts reveals AV node TGFb staining and TGFb activation promotes fibroblast trans-differentiation, a scarring phenotype. Since the uPA/uPAR system plays a role in TGFb activation, this study evaluated whether anti-Ro/La binding to apoptotic cardiocytes via plasmin activation stimulates TGFb and promotes a profibrosing phenotype. Methods and Results: Initial analysis showed increased TGFb in supernatants from co-cultures of healthy cards and apoptotic cards incubated with IgG fractions from mothers whose sera contain anti-Ro/La antibodies and who had a child with CHB (apo-CHB-IgG) compared to co-cultures of healthy cards and apoptotic cards incubated with control IgG (apo-nl-IgG). Using a luciferase bioassay of active TGFb, supernatants from co-cultures of healthy cards and apo-CHB-IgG cards exhibited increased levels of active TGFb compared to those cocultured with apo-nl-IgG cards (511pg/ml CHB-IgG RLU vs 217 nl-IgG RLU; p=0.007; n=7). Abrogation of RLU via anti-TGFb antibody or TGFb inhibitor (SB431542) confirmed TGFb activation was solely due to TGFb. Significantly increased uPA levels (903 pg/ml vs 508 pg/ml; p =0.01; n=5) and uPA activity (0.8 units vs 0.04 units; p=0.005; n=3) were demonstrated in supernatants generated from coculture of healthy cards and apo CHB-IgG cards compared to healthy cards and apo nl-IgG, respectively. To determine whether uPA activity was responsible for TGFb activation, coculture experiments were conducted in which the apo-CHB-IgG cards were treated with anti-uPAR or anti-uPA antibodies or the plasmin inhibitor aprotinin prior to coculturing with healthy cards. In all instances treatments attenuated TGFb activation and uPA activity. To evaluate the profibrotic role of the observed TGFb activation, supernatants from either apo-CHB-IgG or apo-nl-IgG cocultures with healthy cards were applied to serum deprived cardiac fibroblasts. Only supernatants derived from cocultures of healthy cards and apo-CHB-IgG cardiocytes promoted transdifferentiation as evidenced by increased SMAc staining, an effect that was decreased when fibroblasts were treated with supernatants where cocultures were pretreated with uPAR antibodies. Supporting the hypothesis that increased uPA activity is causally related to the pathogenesis of CHB, cord blood samples from 12 of 18 children with CHB exhibited increased uPA activity and uPAR cleavage compared to 4 of 14 non CHB children exposed to maternal anti-Ro antibodies. Conclusions: These data suggest that binding of anti-Ro/La antibodies to apoptotic cardiocytes by virtue of increased uPAR-dependent uPA activity trigger TGFb activation thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar
EMBASE:70380417
ISSN: 0004-3591
CID: 130926

Mutations in LTBP4 cause a syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development

Urban, Zsolt; Hucthagowder, Vishwanathan; Schurmann, Nura; Todorovic, Vesna; Zilberberg, Lior; Choi, Jiwon; Sens, Carla; Brown, Chester W; Clark, Robin D; Holland, Kristen E; Marble, Michael; Sakai, Lynn Y; Dabovic, Branka; Rifkin, Daniel B; Davis, Elaine C
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
PMCID:2775835
PMID: 19836010
ISSN: 1537-6605
CID: 120525