Differential expression and cellular localization of novel isoforms of the tendon biomarker tenomodulin.

Tenomodulin (Tnmd, also called Tendin) is classified as a type II transmembrane glycoprotein and is highly expressed in developing as well as in mature tendons. Along with scleraxis (scx), Tnmd is a candidate marker gene for tenocytes. Its function is unknown, but it has been reported to have anti-angiogenic properties. Results in a knockout mouse model did not substantiate that claim. It has homology to chondromodulin-I. Single nucleotide polymorphisms of TNMD have been associated with obesity, macular degeneration, and Alzheimer's disease in patients. In the present study, three Tnmd isoforms with deduced molecular weights of 20.3 (isoform II), 25.4 (isoform III), and 37.1 (isoform I) kDa were proposed and verified by Western blot from cells with green fluorescent protein-linked, overexpressed constructs, tissue, and by qPCR of isoforms from human tissues and cultured cells. Overexpression of each Tnmd isoform followed by immunofluorescence imaging showed that isoforms I and II had perinuclear localization while isoform III was cytoplasmic. Results of qPCR demonstrated differential expression of each Tnmd isoform in patient's specimens taken from flexor carpi radialis, biceps brachii, and flexor digitorum profundus tendons. Knockdown of Tnmd increased the expression of both scleraxis (scx) and myostatin, indicating a potential negative feedback loop between Tnmd and its regulators. Knockdown of all Tnmd isoforms simultaneously also reduced tenocyte proliferation. I-TASSER protein three-dimensional conformation modeling predictions indicated each Tnmd isoform had different structures and potential functions: isoform 1, modeled as a cytosine methyltransferase; isoform 2, a SUMO-1-like SENP-1 protease; and isoform 3, an α-syntrophin, plextrin homology domain scaffolding protein. Further functional studies with each Tnmd isoform may help us to better understand regulation of tenocyte proliferation, tendon development, response to injury and strain, as well as mechanisms in tendinoses. These results may indicate novel therapeutic targets in specific tenomodulin isoforms as well as treatments for tendon diseases.