tenascin-C Transcripts Research Articles

ACE inhibition modifies exercise-induced pro-angiogenic and mitochondrial gene transcript expression.

Skeletal muscle responds to endurance exercise with an improvement of biochemical pathways that support substrate supply and oxygen-dependent metabolism. This is reflected by enhanced expression of associated factors after exercise and is specifically modulated by tissue perfusion and oxygenation. We hypothesized that transcript expression of pro-angiogenic factors (VEGF, tenascin-C, Angpt1, Angpt1R) and oxygen metabolism (COX4I1, COX4I2, HIF-1α) in human muscle after an endurance stimulus depends on vasoconstriction, and would be modulated through angiotensin-converting enzyme inhibition by intake of lisinopril. Fourteen non-specifically trained, male Caucasians subjects, carried out a single bout of standardized one-legged bicycle exercise. Seven of the participants consumed lisinopril in the 3 days before exercise. Biopsies were collected pre- and 3 h post-exercise from the m. vastus lateralis. COX4I1 (P = 0.03), COX4I2 (P = 0.04) mRNA and HIF-1α (P = 0.05) mRNA and protein levels (P = 0.01) showed an exercise-induced increase in the group not consuming the ACE inhibitor. Conversely, there was a specific exercise-induced increase in VEGF transcript (P = 0.04) and protein levels (P = 0.03) and a trend for increased tenascin-c transcript levels (P = 0.09) for subjects consuming lisinopril. The observations indicate that exercise-induced expression of transcripts involved in angiogenesis and mitochondrial energy metabolism are to some extent regulated via a hypoxia-related ACE-dependent mechanism.

The transcriptional regulator megakaryoblastic leukemia‐1 mediates serum response factor‐independent activation of tenascin‐C transcription by mechanical stress

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies. To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR. We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes. TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

ROCK Controls Matrix Synthesis in Vascular Smooth Muscle Cells

Tenascin-C (TN-C) is an extracellular matrix (ECM) protein expressed within remodeling systemic and pulmonary arteries (PAs), where it supports vascular smooth muscle cell (SMC) proliferation. Previously, we showed that A10 SMCs cultivated on native type I collagen possess a spindle-shaped morphology and do not express TN-C, whereas those on denatured collagen possess a well-defined F-actin stress fiber network, a spread morphology, and they do express TN-C. To determine whether changes in cytoskeletal architecture control TN-C, SMCs on denatured collagen were treated with cytochalasin D, which decreased SMC spreading and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling effectors required for TN-C transcription. Next, to determine whether cell shape, dictated by the F-actin cytoskeleton, regulates TN-C, different geometries of SMCs (ranging from spread to round) were engineered on denatured collagen: as SMCs progressively rounded, ERK1/2 activity and TN-C transcription declined. Because RhoA and Rho kinase (ROCK) regulate cell morphology by controlling cytoskeletal architecture, we reasoned that these factors might also regulate TN-C. Indeed, SMCs on denatured collagen possessed higher levels of RhoA activity than those on native collagen, and blocking RhoA or ROCK activities attenuated SMC spreading, ERK1/2 activity, and TN-C expression in SMCs on denatured collagen. Thus, ROCK controls the configuration of the F-actin cytoskeleton and SMC shape in a manner that is permissive for ERK1/2-dependent production of TN-C. Finally, we showed that inhibition of ROCK activity suppresses SMC TN-C expression and disease progression in hypertensive rat PAs. Thus, in addition to its role in regulating vasoconstriction, ROCK also controls matrix production.

Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial hypertension

Familial forms of human pulmonary arterial hypertension (FPAH) have been linked to mutations in bone morphogenetic protein (BMP) type II receptors (BMPR2s), yet the downstream targets of these receptors remain obscure. Here we show that pulmonary vascular lesions from patients harboring BMPR2 mutations express high levels of tenascin-C (TN-C), an extracellular matrix glycoprotein that promotes pulmonary artery (PA) smooth muscle cell (SMC) proliferation. To begin to define how TN-C is regulated, PA SMCs were cultured from normal subjects and from those with FPAH due to BMPR2 mutations. FPAH SMCs expressed higher levels of TN-C than normal SMCs. Similarly, expression of Prx1, a factor that drives TN-C transcription, was elevated in FPAH vascular lesions and SMCs derived thereof. Furthermore, Prx1 and TN-C promoter activities were significantly higher in FPAH vs. normal SMCs. To delineate how BMPR2s control TN-C, we focused on receptor (R)-Smads, downstream effectors activated by wild-type BMPR2s. Nuclear localization and phosphorylation of R-Smads was greater in normal vs. FPAH SMCs. As well, indirect blockade of R-Smad signaling with a kinase-deficient BMP receptor Ib upregulated TN-C in normal SMCs. Because ERK1/2 MAPKs inhibit the transcriptional activity of R-Smads, and because ERK1/2 promotes TN-C transcription, we determined whether ERK1/2 inhibits R-Smad signaling in FPAH SMCs and whether this activity is required for TN-C transcription. Indeed, ERK1/2 activity was greater in FPAH SMCs, and inhibition of ERK1/2 resulted in nuclear localization of R-Smads and inhibition of TN-C. These studies define a novel signaling network relevant to PAH underscored by BMPR2 mutations.

Tenascin C and annexin II expression in the process of pancreatic carcinogenesis

Tenascin C (TNC) is a component of the provisional extracellular matrix (ECM) that characterizes solid tumours. Cell surface annexin II is a high-affinity receptor for large TNC splice variants. The aim of this study was to analyse whether TNC and annexin II play a role in the development of pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by a rich ECM populated by pancreatic stellate cells, which play a crucial role in pancreatic desmoplasia. The mRNA and protein levels of TNC and of annexin II were analysed in pancreatic tissues by DNA array, quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and immunohistochemistry. TNC large splice variants were detected by RT-PCR. Enzyme linked immunosorbent assay (ELISA) was used to measure TNC levels in serum and culture supernatants. TNC and annexin II mRNA levels were significantly higher in pancreatic cancer tissues than in the normal pancreas. TNC expression was detected with increased frequency in the progression from PanIN-1 lesions to PDAC, and a parallel switch from cytoplasmic to cell surface expression of annexin II was observed. Large TNC transcripts were found in pancreatic cancer and in chronic pancreatitis, but not in the normal pancreas. TNC expression was demonstrated in pancreatic stellate cells, where it could be induced by tumour necrosis factor alpha (TNFalpha), transforming growth factor beta1 (TGF-beta1) and by cancer cell supernatants supplemented with TGF-beta1. In conclusion, the expression of TNC and cell surface annexin II increases in the progression from low-grade PanIN lesions to pancreatic cancer. Pancreatic stellate cells are identified as a source of TNC in pancreatic tissues, possibly under the influence of soluble factors released by the tumour cells.

Cell-adhesive responses to tenascin-C splice variants involve formation of fascin microspikes.

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an "antiadhesive" matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.

Human tenascin-C: identification of a novel type III repeat in oral cancer and of novel splice variants in normal, malignant and reactive oral mucosae.

Tenascin-C is a mosaic, linear glycoprotein that is up-regulated during many normal and pathological processes involving either cell migration or tissue morphogenesis, such as invasion of malignant cells and wound healing. Human tenascin-C contains 8 consecutive type III fibronectin (TNCfn) domains that are involved in alternative splicing and potentially generate a large number of isoforms that code for tenascin-C proteins with subtly different functions. Human tenascin-C splice variants were investigated by RT-PCR in a range of normal and pathological oral mucosal tissues. A novel, 9th human TNCfn domain involved in alternative splicing was identified. It shares 70% nucleic acid and 55% protein sequence homology with chicken TNCfn-ad2. As in avians, this novel repeat was located between TNCfn-B and TNCfn-ad1 and accordingly was designated human TNCfn-ad2. Human TNCfn-ad2 was detected in only 2 of 10 oral cancers. However, TNCfn-ad2 was absent from 40 normal, reactive, pre-malignant and other oral mucosal specimens investigated. Previous studies have described 8 splice variant transcripts for human tenascin-C. By systematic investigation we identified further novel splice variants for human tenascin-C. Furthermore, our results indicate that many potential splice variants probably do not exist in the tissues investigated. Thus, we have demonstrated that human tenascin-C transcripts generate a complex but selected repertoire of different alternative splice products.

Tenascin‐C is associated with early stages of chondrogenesis by chick mandibular ectomesenchymal cells in vivo and in vitro

Tenascin-C is an extracellular matrix protein thought to be involved in skeletogenesis. We have examined the distribution of tenascin-C in the developing chick mandibular arch between stages 18–36, and during in vitro chondrogenesis of mandibular ectomesenchymal cells in micromass cultures using a probe and antibody that correspond to the portion of the tenascin-C transcript conserved in all of the three known chick splice variants. In situ hybridization and immunohistochemical analyses demonstrate that tenascin-C is predominantly expressed in the condensing mesenchyme of developing cartilage, and in the perichondrium of differentiated cartilage. Tenascin-C expression, although detected in differentiating chondroblasts, was not detected in differentiated cartilage. Tenascin-C was also expressed in the developing membranous bones. In addition, the expression of tenascin-C transcripts during in vitro chondrogenesis of mandibular ectomesenchymal cells in micromass cultures was compared to the patterns of expression of aggrecan core protein and α1(I) collagen transcripts. Our in situ hybridization analyses of micromass cultures demonstrate the expression of tenascin-C and agreecan core protein mRNAs by pre-chondrogenic aggregates in the 1-day cultures and by chondroblasts in differentiating cartilage nodules in 2-day cultures. In 4- and 9-day cultures, the pattern of expression of tenascin-C mRNA was different from the patterns of expression of aggrecan core protein mRNA, and appeared to be more closely related to the expression of α1(I) collagen mRNA. Aggrecan core protein mRNA was expressed by chondrocytes in cartilage nodules in 4- and 9-day cultures. On the other hand, tenascin-C and α1(I) collagen mRNAs, in addition to being expressed in the loose connective tissues in the inter-nodular spaces, were predominantly expressed by the elongated, flattened, and fibroblast-like cells around the cartilage nodules. These results indicate that during the in vitro chondrogenesis of mandibular ectomesenchymal cells, expression of tenascin-C mRNA identifies chondrocytes in their early stages of differentiation. The patterns of expression of tenascin-C mRNA in 4- and 9-day cultures further suggest that tenascin-C is expressed in the perichondrium-like structures that form around the cartilage nodules in micromass cultures. Therefore, our in vitro studies, in agreement with our in vivo studies, suggest an association of tenascin-C with the initial or early stages of chondrogenesis in the chicken mandibular arch. © 1996 Wiley-Liss, Inc.

Tenascin-C is associated with early stages of chondrogenesis by chick mandibular ectomesenchymal cells in vivo and in vitro

Tenascin-C is an extracellular matrix protein thought to be involved in skeletogenesis. We have examined the distribution of tenascin-C in the developing chick mandibular arch between stages 18-36, and during in vitro chondrogenesis of mandibular ectomesenchymal cells in micromass cultures using a probe and antibody that correspond to the portion of the tenascin-C transcript conserved in all of the three known chick splice variants. In situ hybridization and immunohistochemical analyses demonstrate that tenascin-C is predominantly expressed in the condensing mesenchyme of developing cartilage, and in the perichondrium of differentiated cartilage. Tenascin-C expression, although detected in differentiating chondroblasts, was not detected in differentiated cartilage. Tenascin-C was also expressed in the developing membranous bones. In addition, the expression of tenascin-C transcripts during in vitro chondrogenesis of mandibular ectomesenchymal cells in micromass cultures was compared to the patterns of expression of aggrecan core protein and alpha 1(I) collagen transcripts. Our in situ hybridization analyses of micromass cultures demonstrate the expression of tenascin-C and aggrecan core protein mRNAs by pre-chondrogenic aggregates in the 1-day cultures and by chondroblasts in differentiating cartilage nodules in 2-day cultures. In 4- and 9-day cultures, the pattern of expression of tenascin-C mRNA was different from the patterns of expression of aggrecan core protein mRNA, and appeared to be more closely related to the expression of alpha 1(I) collagen mRNA. Aggrecan core protein mRNA was expressed by chondrocytes in cartilage nodules in 4- and 9-day cultures. On the other hand, tenascin-C and alpha 1(I) collagen mRNAs, in addition to being expressed in the loose connective tissues in the inter-nodular spaces, were predominantly expressed by the elongated, flattened, and fibroblast-like cells around the cartilage nodules. These results indicate that during the in vitro chondrogenesis of mandibular ectomesenchymal cells, expression of tenascin-C mRNA identifies chondrocytes in their early stages of differentiation. The patterns of expression of tenascin-C mRNA in 4- and 9-day cultures further suggest that tenascin-C is expressed in the perichondrium-like structures that form around the cartilage nodules in micromass cultures. Therefore, our in vitro studies, in agreement with our in vivo studies, suggest an association of tenascin-C with the initial or early stages of chondrogenesis in the chicken mandibular arch.

Distinct tissue distribution in pigs of tenascin-X and tenascin-C transcripts.

Tenascin-X and tenascin-C glycoproteins are phylogenetically conserved components of the extracellular matrix, although their specific roles remain to be determined. cDNA probes were produced from pig tenascin-X and tenascin-C genes and were used to examine the tissue distribution of the transcripts in 28 tissues from Large-White pigs, 4.5-42-months old (called adults) and 17 tissues from 87-day-old fetuses. The hybridization of Northern blots with tenascin-X probes revealed, in most tissues, a complex pattern of bands including a major band of about 13 kb, assumed to correspond to the main tenascin-X transcript. Hybridization with the tenascin-C probe showed two transcripts of 6.8 kb and 8.2 kb. The data from the ribonuclease-protection technique showed that both genes displayed large variations in the transcription levels among the tissues analysed. Overall, the tenascin-X gene was significantly expressed in two thirds of the tissues, and the tenascin-C gene in about 50% of them. The highest tenascin-X signals were observed in tendons, ligaments and, unexpectedly, in peripheral nerves. Other tissues, including colon, dermis, skin, heart, uterus, stomach, jejunum, placentae, aorta, lung, mammary and adrenal glands also exhibited significant signal intensities. In fetuses, mainly testes and skeletal muscle showed higher transcription levels than the adult counterparts. The tenascin-C gene was predominantly transcribed in the ligament, tendon, adrenal gland and colon, and more weakly in the stomach, jejunum, lung and spinal cord. In fetuses, the tenascin-C signal in the brain was higher than the signal in the brain of adult, whereas the reverse was true for the adrenal gland and the colon. Within a given tissue, the level of tenascin-X and tenascin-C transcripts varied greatly, indicating independent tenascin-X and tenascin-C transcription regulation mechanisms; this was particularly obvious in adult and fetal nerves but also in the dermis, skin, heart, uterus, placentae and aorta, where tenascin-X RNA molecules were much more abundant than those of tenascin-C. In addition, similar differences were observed in the skeletal muscle and adrenal gland of fetuses. In contrast, the amount of tenascin-C transcripts in the fetal brain and adult spinal cord was higher than those for tenascin-X. Our results draw attention to a possible specific role of tenascin-X in the peripheral nerve physiology.

Syndecan‐3, tenascin‐C, and the development of cartilaginous skeletal elements and joints in chick limbs

The mechanisms by which the early limb cell condensations and interzone mesenchyme give rise to skeletal elements and joints are poorly understood. Previous work from this laboratory has shown that the extracellular matrix protein tenascin-C is associated with articular cartilage and joint tissue development; others have shown that tenascin-C may exert its biological activities via interactions with cell surface receptors, such as syndecans. To further analyze the roles of tenascin-C and its putative receptors in skeletal development, we carried out a detailed in situ hybridization analysis of tenascin-C and syndecan-3 gene expression during development of chick limb skeletal elements and joints. We found that as the early mesenchymal condensations chondrify around day 5 (E5) of development, they become surrounded by a thick syndecan-3 rich perichondrium while tenascin-C transcripts are much fewer and restricted to diaphyseal perichondrium and developing interzones. Similar patterns were observed as distal carpal and digit condensations formed in older embryos. As the cartilaginous long bone models elongated proximo-distally and joint formation proceeded with age, we observed that syndecan-3 transcripts decrease significantly along the diaphysis and remain very abundant along the metaphysis and in the epiphyseal articular cap and interzone. Conversely, tenascin-C RNAs remain abundant along the diaphysis and begin to increase at the epiphysis and in interzone-derived tissues, such as menisci and joint capsule. By E10, the skeletal elements have well-defined morphologies, endochondral ossification has initiated in their diaphysis, and diaphyseal perichondrium has become periosteum. These developmental changes were accompanied by equally marked changes in gene expression; these included a marked increase in tenascin-C gene expression in articular cap, fragmentation of tenascin-C gene expression along the periosteum, reinitiation of syndecan-3 gene expression in periosteum, and differential gene expression in osteoprogenitor cells. The sheer complexity of the gene expression patterns documented in this study attests to the complexity of processes that bring about normal skelatogenesis. Clearly, tenascin-C and syndecan-3 appear to be closely associated with several of these processes, particularly in establishing tissue boundaries (perichondrium and periosteum) between condensations and surrounding mesenchymal cells, in regulating perichondral cell differentiation and incorporation into the growing skeletal elements, and in the genesis of epiphyseal chondrocytes and associated joint tissues.