Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family (original) (raw)
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Development, 2009
Tendons and ligaments mediate the attachment of muscle to bone and of bone to bone to provide connectivity and structural integrity in the musculoskeletal system. We show that TGFβ signaling plays a major role in the formation of these tissues. TGFβ signaling is a potent inducer of the tendon progenitor (TNP) marker scleraxis both in organ culture and in cultured cells, and disruption of TGFβ signaling in Tgfb2 -/-;Tgfb3 -/double mutant embryos or through inactivation of the type II TGFβ receptor (TGFBR2; also known as TβRII) results in the loss of most tendons and ligaments in the limbs, trunk, tail and head. The induction of scleraxis-expressing TNPs is not affected in mutant embryos and the tendon phenotype is first manifested at E12.5, a developmental stage in which TNPs are positioned between the differentiating muscles and cartilage, and in which Tgfb2 or Tgfb3 is expressed both in TNPs and in the differentiating muscles and cartilage. TGFβ signaling is thus essential for maintenance of TNPs, and we propose that it also mediates the recruitment of new tendon cells by differentiating muscles and cartilage to establish the connections between tendon primordia and their respective musculoskeletal counterparts, leading to the formation of an interconnected and functionally integrated musculoskeletal system.
Developmental Dynamics, 2008
Tendon is one of the least understood tissues of the musculoskeletal system in terms of development and morphogenesis. Collagen fibrillogenesis has been the most studied aspect of tendon development, focusing largely on the role of matrix molecules such as collagen type III and decorin. While involvement of matrix molecules in collagen fibrillogenesis during chick tendon development is well understood, the role of growth factors has yet to be elucidated. This work examines the expression patterns of TGF-ß1, -ß2, and -ß3, and their receptors with respect to expression patterns of collagen type III, decorin, and fibronectin. We focus on the intermediate stages of tendon development in the chick embryo, a period during which the tendon micro-and macro-architecture are being established. Our findings demonstrate for the first time that TGF-ß1, -ß2, and -ß3 have distinct spatiotemporal developmental protein localization patterns in the developing tendon and strongly suggest that these isoforms have independent roles in tendon development.
Effect of TGF-β inducible early gene deficiency on flexor tendon healing
Journal of Orthopaedic Research, 2006
The role of transforming growth factor b (TGF-b) in tendon healing is still not clearly established. TGF-b affects gene expression primarily through the activation of the Smad signaling pathway. The first step in the Smad pathway is the expression of TGF-b inducible early gene (TIEG). Recently, a TIEG knockout mouse has been developed. The purpose of this study was to examine the healing potential of flexor tendons in mice lacking the TIEG gene, and to further examine what role the TIEG pathway plays in flexor tendon repair. Twenty-two mice, consisting of 11 normal wild-type mice and 11 TIEG knockout mice, were euthanized at 8 to 12 weeks of age. The second through fifth FDL tendons of both hind feet were transected and repaired in zone 2. The repaired tendons were removed from the mice and placed into tissue culture. Tendons were then examined at days 3, 7, 14, 21, and 42 after surgery. Hematoxylin and eosin (HE) staining and immunohistochemical staining for TGF-b, collagen type I, and collagen type III were performed. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to examine expression of TGF-b1, b2, b3, and collagen type I and III. At 42 days after surgery, HE staining showed coaptation of lacerated tendon ends in both groups. Both groups showed healing of the lacerated tendon, but the chronologic expression pattern of TGF-b was different between the knockout and normal tendons. TIEG deficient tendons had delayed expression of TGF-b when compared with control tendons. The collagen mRNA expression pattern was similar with both groups, but the expression level was different, with TIEG knockout tendons having a lower expression of collagen type I mRNA (p < 0.001). TGF-b is thought to play a major role in tendon healing. Healing of tendons in the TIEG knockout mouse suggests the possibility of tendon healing in the absence of the Smad pathway. The knockout mouse model described in the present study provides a novel means for further understanding of the tendon healing process through isolated deletion of specific growth factors. ß
In Vitro Flexor Tendon Cell Response to TGF-β1: A Gene Expression Study
The Journal of Hand Surgery, 2009
Purpose Adhesion formation around zone II flexor tendon repairs remains an important clinical challenge. Tendon healing is complex, and when uncontrolled it may lead to adhesion formation. Transforming growth factor-1 (TGF-1) is a multipotent growth factor known to be involved in wound healing and scar formation. It has also been shown to have a role in both tendon healing and adhesion formation. Methods Uninjured rabbit flexor tendons were divided into endotenon, epitenon, and sheath cells and cultured separately. The in vitro effect of TGF-1 gene expression was determined on quiescent tendon cells using real-time polymerase chain reaction for collagen type 1, collagen type 3, fibronectin, plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (t-PA). Results Endotenon-derived cells showed a statistically significant down-regulation of collagen type I gene expression in response to TGF-1 compared with untreated endotenon cells and with both epitenon and sheath cells at a number of time points. However, endotenon cells showed an increase in collagen type 3 gene expression compared with untreated cells and epitenon cells. All cells showed a statistically significant increase in fibronectin in the later time points compared with the untreated cells. Endotenon-derived cells showed an early increase in PAI-1, whereas sheath cells showed a later increase. Conclusions We have shown that cells cultured from 3 separate parts of the flexor tendon-sheath complex respond in different ways when stimulated with TGF-1. The down-regulation of collagen types 1 and 3 in endotenon cells may give further insight into the effects of TGF-1 in tendon healing. Also, the upregulation of fibronectin and PAI-1, combined with a downregulation of tissue plasminogen activator, could explain the association of TGF-1 with tendon adhesion formation. Treatments aimed at improving tendon healing and the prevention of adhesions may arise from modification of the effects of TGF-1.
Preferential tendon stem cell response to growth factor supplementation
Tendon injuries are increasingly prevalent around the world, accounting for more than 100 000 new clinical cases/year in the USA alone. Cell-based therapies have been proposed as a therapeutic strategy, with recent data advocating the use of tendon stem cells (TSCs) as a potential cell source with clinical relevance for tendon regeneration. However, their in vitro expansion is problematic, as they lose their multipotency and change their protein expression profile in culture. Herein, we ventured to assess the influence of insulin-like growth factor 1 (IGF-1), growth and differentiation factor-5 (GDF-5) and transforming growth factor-β1 (TGFβ1) supplementation in TSC culture. IGF-1 preserved multipotency for up to 28 days. Upregulation of decorin and scleraxis expression was observed as compared to freshly isolated cells. GDF-5 treated cells exhibited reduced differentiation along adipogenic and chondrogenic pathways after 28 days, and decorin, scleraxis and collagen type I expression was increased. After 28 days, TGFβ1 supplementation led to increased scleraxis, osteonectin and collagen type II expression. The varied responses to each growth factor may reflect their role in tendon repair, suggesting that: GDF-5 promotes the transition of tendon stem cells towards tenocytes; TGFβ1 induces differentiation along several pathways, including a phenotype indicative of fibrocartilage or calcified tendon, common problems in tendon healing; and IGF-1 promotes proliferation and maintenance of TSC phenotypes, thereby creating a population sufficient to have a beneficial effect.
Journal of Biological Chemistry, 2009
Transforming growth factor  (TGF) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGF signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFs shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved downregulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGF signaling up-regulates Sox9 in the in vivo experimental model system in which TGF treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGF signaling, we found that TGFs appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGF signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGF signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGF treatments.
Development (Cambridge, England), 2016
The molecular program underlying tendon development is not fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles, however muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, while the FGF/ERKMAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERKMAPK and TGFβ/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFβ2 ligands prevents SCX downregulation in limbs in immobilisation conditions. TGFβ2 but not FGF4 prevent TNMD and THBS2 downregulation in immobilisation conditions. We ...
Annals of the Rheumatic Diseases, 2006
To investigate effects of cartilage derived morphogenetic protein-1 and-2 (CDMP-1, CDMP-2), bone morphogenetic protein (BMP)-7 and BMP-6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential. Methods: Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP-1, CDMP-2, BMP-7, and BMP-6. Expression of the indicated growth factors was assessed by RT-PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT-PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [ 35 S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures. Results: CDMP-1, CDMP-2, BMP-7, and BMP-6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [ 35 S]sulphate incorporation. Toluidine blue staining showed cartilage-specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers. Conclusion: CDMP-1, CDMP-2, BMP-7, and BMP-6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP-1, CDMP-2, BMP-7, and BMP-6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair.
TGF-β signaling is critical for maintenance of the tendon cell fate
Studies of cell fate focus on specification, but little is known about maintenance of the differentiated state. We find that TGFβ signaling plays an essential role in maintenance of the tendon cell fate. To examine the role TGFβ signaling in tenocytes TGFb type II receptor was targeted in the Scleraxis cell lineage. Tendon development was not disrupted in mutant embryos, but shortly after birth tenocytes lost differentiation markers and reverted to a more stem/progenitor state. Targeting of Tgfbr2 using other Cre drivers did not cause tenocyte dedifferentiation suggesting a critical significance for the spatio-temporal activity of ScxCre. Viral reintroduction of Tgfbr2 to mutants was sufficient to prevent and even rescue mutant tenocytes suggesting a continuous and cell-autonomous role for TGFβ signaling in cell fate maintenance. These results uncover the critical importance of molecular pathways that maintain the differentiated cell fate and a key role for TGFβ signaling in these p...
The role of connective tissue growth factor in skeletal growth and development
Medical science monitor : international medical journal of experimental and clinical research, 2006
Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated protein that regulates diverse cellular functions in different cell types. CTGF gene belongs to a larger CCN gene family that also includes Cyr61 and NOV. It modulates many cellular functions, including proliferation, migration, adhesion, and extracellular matrix production, and it is involved in many biological and pathological processes. CTGF has special importance in skeletal development. During Meckel's cartilage development, CTGF acts as a down-stream molecule of TGFbeta to stimulate cell-cell interactions and the expression of condensation-associated genes. CTGF promotes endochondral ossification and articular cartilage regeneration. During the healing of experimental bone fracture, CTGF was expressed in periosteal cells and hypertrophic chondrocytes. It promotes the proliferation of chondrocytes and osteoblasts. CTGF is a down-stream mediator for prostaglandin E2 (PGE2) in osteoblast-ind...