Spatiotemporal protein distribution of TGF-βs, their receptors, and extracellular matrix molecules during embryonic tendon development (original) (raw)
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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 ...
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.
Clinical orthopaedics and related research, 2005
Transforming growth factor-beta1 is known for its effect on the production of extracellular matrix in tendons. Elevated levels of transforming growth factor-beta1 have been reported in tendon adhesion and tendinosis, which suggests that transforming growth factor-beta1 plays an important role in matrix disturbances. Tendon adhesion involves excessive collagen deposition, whereas tendinosis is associated with increased proteoglycan deposition. It seems that other factors also may affect matrix deposition and modulate the effects of transforming growth factor-beta1. We assessed whether matrix anchorage to Type I collagen or fibronectin could change the gene expression of matrix proteins in tendon fibroblasts, and studied whether the effects of transforming growth factor-beta1 were altered by matrix anchorage. Human patellar tendon fibroblast cultures were prepared in different cell anchorages, and the cellular responses to transforming growth factor-beta1 were measured as gene express...
Journal of Hand Surgery, 2004
This study analysed the differences on a molecular level between two segments of the deep flexor tendon, and compared the intrasynovial flexor tendon with the tendon sheath and the extrasynovial peroneus tendon in a rabbit model. The TRIspin method of RNA extraction was combined with the reverse transcription polymerase chain reaction to assess mRNA levels in the tissue segments. Significant differences were detected for all genes studied. mRNA levels for aggrecan, biglycan and collagen III were significantly higher in the fibrocartilaginous proximal segment of the flexor tendon. Collagen I was higher in the flexor tendon than the sheath and the peroneus tendon, and TGF-β1 was significantly lower in the peroneus tendon. This study demonstrates differences at the mRNA level between different segments of tendon, indicating that the tendon tissue may be adapted to its environment.
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.
Inhibition of TGF-β-induced collagen production in rabbit flexor tendons1
Journal of Hand Surgery-american Volume, 2004
Purpose: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF- neutralizing antibody in blocking TGF--induced collagen I production in rabbit flexor tendons in vitro. Methods: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF- along with increasing doses of TGF- neutralizing antibody (0.1-2.0 g/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF- bioactivity was measured by the luciferase assay. Results were compared with TGF- alone and unsupplemented controls. Results: The addition of neutralizing antibody significantly reduced TGF--induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF- bioactivity was also reduced by its neutralizing antibody. Conclusions: This study shows that TGF- inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF- on flexor tendons clinically. (J Hand Surg 2004;29A:230 -235.
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...
Inhibition of TGF-β-induced collagen production in rabbit flexor tendons
The Journal of Hand Surgery, 2004
Purpose: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF- neutralizing antibody in blocking TGF--induced collagen I production in rabbit flexor tendons in vitro. Methods: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF- along with increasing doses of TGF- neutralizing antibody (0.1-2.0 g/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF- bioactivity was measured by the luciferase assay. Results were compared with TGF- alone and unsupplemented controls. Results: The addition of neutralizing antibody significantly reduced TGF--induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF- bioactivity was also reduced by its neutralizing antibody. Conclusions: This study shows that TGF- inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF- on flexor tendons clinically. (J Hand Surg 2004;29A:230 -235.
Journal of Clinical Investigation, 1997
Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF- gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis. ( J.
Journal of musculoskeletal & neuronal interactions, 2005
Weight-bearing tendons in many species, including humans, chickens and horses, are prone to failure, in many cases without a discernible cause. The normal function of the tendon depends on the proper assembly of fibrils of type I collagen, the main structural component of the tendon. We studied the effect of in vitro culture, temperature (37 degrees C vs. 43 degrees C) and wounding on the expression of mRNAs for several collagen regulators, transforming growth factor beta (TGF(beta)), heat shock protein 47 (Hsp47) and connective tissue growth factor (CTGF), in chicken embryonic gastrocnemius tendon explants. The expression of mRNAs for TGF(beta) and Hsp47, a chaperone of collagen assembly, remained strong during the first day of in vitro culture, but then it decreased, slightly more at higher temperature. Additional injury in selected tendons had no significant effect on the levels of TGF(beta) and Hsp47 mRNAs. Likewise, the level of immunostained type I procollagen also decreased w...