Table 2.
Summary of the discovered functions of Tnmd.
| Reference | Experimental model | Study type | Findings and functions of Tnmd |
|---|---|---|---|
| Brandau et al., 2001 | Northern blot analysis of new born mouse RNA; ISH on E17.5 mouse embryos and adult mouse brain; cloning of human and mouse Tnmd cDNA; genomic analysis of human Tnmd organization | Genomic study and in vivo | - Strong expression of 1.5 kb Tnmd transcript in diaphragm, skeletal muscle and eye; low expression in brain, liver, lung, kidney, heart, thymus and perichondrium- and periosteum-free ribcage; Tnmd transcript found in tendons and ligaments but also in the brain, spinal cord, liver, lungs, bowel, thymus and eye of E17.5 mouse embryos; high expression of Tnmd mRNA in the dentate gyrus, CA regions of the hippocampus, neurones in the cerebral nuclei, Purkinje cells and neuronal cells in the cerebellar nucleus, neurons in the anterior and posterior horn of the spinal cord and neurons from all cell layers in the cerebral cortex of adult mice. - Mouse and human Tnmd share 89% homology, similarity (54%) and identity (31%) to Chm1, especially with the C-terminal (77% and 66%); 7 exons found. |
| Shukunami et al., 2001 | Cloning of mouse Tnmd; Northern blot analysis of mouse tissues; ISH of mouse skeletal muscle | Genomic study and in vivo | - Cloning of Tnmd full-length reveals novel protein of 317 amino acid residues; predicted amino acid sequence revealed 33% overall identity with mouse Chm1 precursor; structural features include singly transmembrane domain at the N-terminal region and the putative angiogenic domain with eight cysteine residues; Tnmd lacked a hormone-processing signal compared to Chm1, suggesting it may function as a type II transmembrane protein on cell surface. - Tnmd transcript of 1.4 kb detected in skeletal muscle; Tnmd expression not associated with muscle fibres, but rather with epimysium and tendon. |
| Oshima et al., 2003 | Northern blot analysis on mouse embryos; ISH on eyes of mouse embryos; human retinal endothelial cells (HRECs) transduced with Tnmd; human umbilical vein endothelial cells (HUVECs) co-cultured with HRECs transduced with Tnmd | In vivo and in vitro | -Tnmd mRNA expression detectable from day E15.5 and present in eye and skin; localization on Tnmd transcript on the extraocular muscle, sclerocornea, lens fibre cells, ganglion cell layer, inner nuclear layer cells and pigment epithelium of the retina. - Effective autocrine suppression of cell proliferation and capillary-like morphogenesis of retina vascular endothelial cells; conditioned media from soluble Tnmd-overexpressing cells also showed marked inhibitory effect on angiogenesis. |
| Oshima et al., 2004 | Adenoviral expression system to force expression of Tnmd C-Terminal in HUVECs; C57BL/6 mouse model subcutaneously injected with melanoma tumour cells; histological analysis of melanoma tumour cells injected into mice; transduction of Tnmd into BL-6 melanoma cells | In vivo and in vitro | - Transduction of Tnmd into HUVECS impaired tube formation of HUVECs cultured in Matrigel; conditioned media from COS7 cells transfected with Tnmd impaired tube formation of HUVECs; transformation of HUVECS with Tnmd downregulated VEGF synthesis to 4-50% of normal levels; migration of HUVECs in response to VEGF was significantly affected after Tnmd transduction. - Formed tumours were 46% smaller compared to Ad-EGFP-transduced melanoma cells; visible inhibition of angiogenesis in implanted tumours and decreased microvessel density; no effect on growth rate. |
| Pisani et al., 2004 | Northern blot analysis on gastrocnemius muscle of rats; RT-PCR analysis of tendons separated from soleus muscle and tendon-free soleus muscle of rats and Tnmd in primary human muscle cells derived from single satellite cells; quantitative Northern blot analysis of C2C12 mouse cell line; overexpression of Tnmd fused to FLAG peptide and immunofluorescence to FLAG; Western blot analysis of myodulin-FLAG fusion protein; co-culture experiment of C2C12 mouse myoblast with H5V cells; proliferation of WT muscle cells and Tnmd-FLAG C2C12 cells compared; Tnmd expression in hind limb suspension model in rats | In vivo and in vitro | -Tnmd mRNA found in muscle fibres and tendons; Tnmd transcript found at the myoblast proliferating stage and myotube differiated stage; Tnmd transcript found in C2C12 cells without any significant difference in expression levels between proliferating and differentiated stages; evidence of a muscle cell surface protein. - Tnmd-FLAG fusion protein = 44 kDa, FLAG = 1kDa so mass of Tnmd = 43 kDa.Calculated mass = 37.047. - Tnmd has an active role in invasive action of endothelial cells, without evidence of extracellular Tnmd secretion; no differences observed in proliferative capacity between WT muscle cells and Tnmd-FLAG C2C12 cells, even with the addition of FGF-2; soleus muscle mass decrease resulted in capillary loss resulting in three-fold Tnmd downregulation and 2-fold upregulation the in opposite condition. |
| Docheva et al., 2005 | Generation of the Tnmd KO mouse model; Northern blot analysis of Tnmd in new-born thymus, eye, tendon, muscle and heart tissues; oxygen-induced retinopathy model (OIR); electron microscopy of Achilles tendons; immunohistochemical analysis of collagens, decorin, lumican, aggrecan and matrilin-2 | In vivo | - Successful generation of Tnmd KO mouse line validated by lack of Tnmd transcript; high Tnmd expression in tendons and at lower levels in eye and muscle. - Probing of tendon extracts with an antibody raised against the C-terminal domain of Tnmd, detected a protein signal of 16 kDa corresponding to the cleavable C-terminal cysteine-rich extracellular domain; suggestion that C-terminal cysteine rich domain of Tnmd is rapidly cleaved in vivo - No changes in retinal vascularization seen in both genotypes after OIR. - Loss of Tnmd expression resulted in decreased tenocyte proliferation and reduced tenocyte density in vivo. - No changes in extracellular matrix protein production like collagen type I, II, III and VI and decorin, lumican, aggrecan and matrilin-2; collagen fibrils showed significantly increased average diameters |
| Pisani et al., 2005 | Yeast transformation with mouse Tnmd vector including C-terminal region and Western blotting; purification of Tnmd in yeast by column purification; co-culturing C2C12 and H5V cells together with modified yeast | In vitro | - Mouse Tnmd could be expressed at the plasma membrane of Saccharomyces cervisiae in an N-glycosylated state. - Murine Tnmd can be purified from yeast; detection of three different bands of which one shows Tnmd in a glycosylated state (65 kDa). - Mouse Tnmd expressed in yeast fully functional by increasing invasive potential of C2C12 cells in H5V co-culture system. |
| Shukunami et al., 2006 | ISH on developing chick embryos; retroviral expression of Scx in chick tenocytes and chondrocytes; misexpression of RCAS-cScx by electroporation into hind limb | In vivo and in vitro | - At stage 23, Scx expression in the syndetome has extended to the tail region and Tnmd detectable in anterior eight somites; at stage 25, Tnmd and Scx detectable in regions adjacent to the myotome; at stage 32 and later, Scx and Tnmd displayed similar expression profiles in developing tendons. - Upregulation of Tnmd in tenocytes but not chondrocytes; no induction of generation of additional tendons but resulted in upregulation of Tnmd in tendons at stage 33 and later. |
| Watahiki et al., 2007 | Quantitative PCR of mandibular condylar cartilage and tibial growth plate cartilage of 1-and 5 week old rats; IHC analysis on mandibular-, condylar- and tibial cartilage of 1-and 5- week old rats | In vivo | - Mandibular condylar cartilage showed higher mRNA levels of Tnmd than tibial growth plate cartilage; mandibular condylar cartilage 1-week-old rats presented higher Tnmd mRNA levels than 5-weeks-old rats; Tnmd was found on condylar cartilage except on fibrous layer; Tnmd only detected in the hypertrophic layer and part of articular cartilage. |
| Shukunami et al., 2008 | In situ hybridization on developing mouse at E14.5; immunohistochemical analysis on developing mouse forelimb; ISH on avascular mesenchyme and vertebrae of developing chick | In vivo | - mRNA localized in developing tendons which were co-localized to Scx expression; Tnmd staining spotted in cartilage and localization restricted to the PECAM-1 negative region of the developing tendons; first identified in posterior regions of limb mesenchyme at HH stage 23 and later expressed in attachment sites connecting cartilage and muscle; at HH stage 30, distinct expression pattern adjacent to MyoD-positive domain marking the myotome and partially overlapping with Scx expression; Tnmd-positive domains were lacking blood vessels; Tnmd expression exclusive to connective tissues including intervertebral region, perichondrium of vertebrae and ligaments; Tnmd only identified in peripheral region where thick bundles of collagen fibers were present. |
| Wang et al., 2012 | OIR in mice; injection of commercially produced recombinant Tnmd into eye for analysis of blood vessel patterns | In vivo | - In OIR model, fewer central non-perfused areas were observed in Tnmd-injected eyes than in PBS-injected eyes; significantly less nuclei in new blood vessels breaking in each retinal cross-section of Tnmd-treated- compared to PBS-treated mice; suggestion of potential role of Tnmd in prevention and treatment of ocular neovascularization. |
| Qi et al., 2012 | I-TASSER protein three-dimensional conformation modeling prediction; human and porcine tenocytes; knock- down of human FCR cells; Tnmd Western blotting of pig, horse and human tendon cells; ICC on Tnmd isoforms overexpressed in COS-7 cells | Computer analysis and in vitro | - Three TNMD transcripts with molecular weights of 37.1, 20.3 and 25.4 kDa for isoforms I, II and II respectively proposed and verified by Western blot from human cells; isoforms I and II localized perinuclear and isoform III on cytoplasm; predicted function of TNMD I = cytosine methyltransferase, II= SUMO--1-like SENP-1 protease, III= α-syntrophin, pleckstrin homology domain scaffolding protein. - Knockdown of all isoforms resulted in reduced cell proliferation and downregulation of myostatin and scleraxis. |
| Komiyama et al., 2013 | Tnmd IHC on WT mouse molars; FL-, CTD-, CS-, BRICHOS-, and EC Tnmd overexpression in NIH3T3 and hPDL cells; cleavage of N-glycans and inhibition of glycosylation in vitro analysis by adding tunicamycin and digesting cell lysates with PNGaseF respectively; analysis of adhesion capacity of Tnmd KO mouse fibroblasts | In vivo and in vitro | -Tnmd expression is related to time of tooth eruption at 2-3 weeks of age; Tnmd localized in Golgi apparatus, microtubules and plasma membrane of PDL cells. - 45-kDa Tnmd protein is likely to be glycosylated form; 40-kDa protein the non-glycosylated Tnmd protein. - Tnmd overexpression resulted in in enhanced adhesion of PDL and NIH3T3 cells to collagen I and increased surface area; Tnmd KO fibroblasts showed decreased cell adhesion; BRICHOS domain or CS region seem responsible for Tnmd-mediated improvement of cell adhesion in Tnmd overexpressing NIH3T3 cells. |
| Miyabara et al., 2014 | Wnt in vitro model; equine BMSCs cultured in collagen gel; nuclear translocation of ß-catenin in glycogen synthase kinase-3 inhibitor-treated BMSCs | In vitro | - mRNA expression of Tnmd in equine BMSCs increased when Wnt pathway was activated, comparable to levels in tendon. - Concluded regulation of tenomodulin expression via Wnt/ß-catenin signaling. |
| Sato et al., 2014 | Tnmd messenger analysis using qPCR of masseter muscle (MM) from C57/BL6 mice; ISH of Tnmd transcript MM | In vivo | - Level of Tnmd RNA increased from E12.5-E17.5 and decreased after birth; CD31 and VEGF mRNA levels in the MM remained constant from E12.5-E18.5 and low after birth; Tnmd mRNA found in the middle region of the MM at E12.5 as well as in the muscle-bone junction from E14.5 onwards. |
| Alberton et al. 2015 | Self-renewal comparison between WT and Tnmd KO TSPC; transfection of full-length and C-terminal domain of Tnmd in Tnmd KO mouse TSPCs; multipotential comparison on TSPCs | In vitro | - Tnmd KO TSPCs show reduced proliferative capacity. - C-terminal alone restores proliferation in Tnmd KO TSPCs. - Loss of Tnmd results in premature and increased senescence of TSPCs compared to WT. - Lack of Tnmd does not affect multipotential in TSPC. |
Abbreviations: Ad-EGFP, adenovirus vector encoding enhanced green fluorescent protein; BMSCs, bone marrow stromal cells; C2C12, mouse myoblast cell line; C57BL/6, C57 black 6; CA region, Cornu Ammonis region; cDNA, complementary deoxyribonucleic acid; Chm1; chondromodulin-1; COS-7, African green monkey fibroblast-like kidney cell line; CS, mutant with deleted cleavage site; CTD, C-terminal domain deletion mutant; E, embryonic day; EC domain, mutant with entire extracellular portion of Tnmd deleted; FCR, flexor carpi radialis; FGF-2, fibroblast growth factor-2; FL, full length Tnmd; H5V, mouse embryonic heart endothelial cells; HH, Hamburger-Hamilton stage; hPDL, human periodontal ligament; ICC, immunocytochemistry; IHC, immunohistochemistry; ISH, in situ hybridization; I-TASSER, iterative, threading assembly refinement; kb, kilo base; kDa, kilodalton; KO, knockout; mRNA, messenger ribonucleic acid; NIH3T3, mouse embryonic fibroblasts; OIR, oxygen-induced retinopathy; PBS, phosphate-buffered saline; PDL, periodontal ligament; PNGaseF, peptide-N-glycosidase F; qPCR, quantitative PCR; RCAS-cScx, replication-competent avian sarcoma-leukosis virus-copy Scleraxis; RNA, ribonucleic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; Scx, Scleraxis; SUMO, small ubiquitin-like modifier; Tnmd, tenomodulin; VEGF, vascular endothelial growth factor; WT, wildtype.