Abstract
Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5′-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (−1030 to −295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5′-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.
Highlights
Human and mouse tenomodulin (Tnmd) are type II transmembrane glycoproteins consisting of 317 amino acids[1,2]
In human umbilical vein endothelial cells (HUVECs), proliferation, migration, adhesion to vitronectin, and tube formation on Matrigel were significantly inhibited by adenoviral overexpression of mature Chmd or by overexpression of the C-terminal 116-amino acid fragment of Tnmd containing a secretion signal sequence[6,7]
Tnmd expression in chick leg tendons is positively regulated by scleraxis (Scx), a basic helix-loop-helix transcription factor that marks both tendon progenitors and tenocytes during chick and mouse embryonic development[8]
Summary
Human and mouse tenomodulin (Tnmd) are type II transmembrane glycoproteins consisting of 317 amino acids[1,2]. Double immunostaining of CD31, a cell surface marker of vascular endothelial cells, and Tnmd or Chmd in the mouse forelimb at embryonic day (E) 16.5 revealed that Tnmd and Chmd proteins localize to the avascular region of tendon and cartilage, respectively[9]. Both Tnmd and Chmd are anti-angiogenic molecules expressed in association with avascular mesenchymes. Scx directly transactivates Tnmd via these E-box sites to positively regulate tenocyte/ligamentocyte maturation during development and growth
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