Expression analysis of α-smooth muscle actin and tenascin-C in the periodontal ligament under orthodontic loading or in vitro culture.

α-smooth muscle actin (α-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of α-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-ß1 (TGF-ß1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of α-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in α-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of α-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.

Inhalation therapy with the synthetic TIP-like peptide AP318 attenuates pulmonary inflammation in a porcine sepsis model.

BACKGROUND: The lectin-like domain of TNF-α can be mimicked by synthetic TIP peptides and represents an innovative pharmacologic option to treat edematous respiratory failure. TIP inhalation was shown to reduce pulmonary edema and improve gas exchange. In addition to its edema resolution effect, TIP peptides may exert some anti-inflammatory properties. The present study therefore investigates the influence of the inhaled TIP peptide AP318 on intrapulmonary inflammatory response in a porcine model of systemic sepsis. METHODS: In a randomized-blinded setting lung injury was induced in 18 pigs by lipopolysaccharide-infusion and a second hit with a short period of ventilator-induced lung stress, followed by a six-hour observation period. The animals received either two inhalations with the peptide (AP318, 2×1 mg kg(-1)) or vehicle. Post-mortem pulmonary expression of inflammatory and mechanotransduction markers were determined by real-time polymerase chain reaction (IL-1ß, IL-6, TNF-α, COX-2, iNOS, amphiregulin, and tenascin-c). Furthermore, regional histopathological lung injury, edema formation and systemic inflammation were quantified. RESULTS: Despite similar systemic response to lipopolysaccharide infusion in both groups, pulmonary inflammation (IL-6, TNF-α, COX-2, tenascin-c) was significantly mitigated by AP318. Furthermore, a Western blot analysis shows a significantly lower of COX-2 protein level. The present sepsis model caused minor lung edema formation and moderate gas exchange impairment. Six hours after onset pathologic scoring showed no improvement, while gas exchange parameters and pulmonary edema formation were similar in the two groups. CONCLUSION: In summary, AP318 significantly attenuated intrapulmonary inflammatory response even without the presence or resolution of severe pulmonary edema in a porcine model of systemic sepsis-associated lung injury. These findings suggest an anti-inflammatory mechanism of the lectin-like domain beyond mere edema reabsorption in endotoxemic lung injury in vivo.

Association of the NPAS3 gene and five other loci with response to the antipsychotic iloperidone identified in a whole genome association study.

A whole genome association study was performed in a phase 3 clinical trial conducted to evaluate a novel antipsychotic, iloperidone, administered to treat patients with schizophrenia. Genotypes of 407 patients were analyzed for 334,563 single nucleotide polymorphisms (SNPs). SNPs associated with iloperidone efficacy were identified within the neuronal PAS domain protein 3 gene (NPAS3), close to a translocation breakpoint site previously observed in a family with schizophrenia. Five other loci were identified that include the XK, Kell blood group complex subunit-related family, member 4 gene (XKR4), the tenascin-R gene (TNR), the glutamate receptor, inotropic, AMPA 4 gene (GRIA4), the glial cell line-derived neurotrophic factor receptor-alpha2 gene (GFRA2), and the NUDT9P1 pseudogene located in the chromosomal region of the serotonin receptor 7 gene (HTR7). The study of these polymorphisms and genes may lead to a better understanding of the etiology of schizophrenia and of its treatment. These results provide new insight into response to iloperidone, developed with the ultimate goal of directing therapy to patients with the highest benefit-to-risk ratio.

A pilot study: 131I-antitenascin monoclonal antibody 81c6 to deliver a 44-Gy resection cavity boost.

The purpose of this study was to determine the feasibility and assess the efficacy and toxicity, among newly diagnosed malignant glioma patients, of administering (131)I-labeled murine antitenascin monoclonal antibody 81C6 ((131)I-81C6) into a surgically created resection cavity (SCRC) to achieve a patient-specific, 44-Gy boost to the 2-cm SCRC margin. A radioactivity dose of (131)I-81C6 calculated to achieve a 44-Gy boost to the SCRC was administered, followed by conventional external beam radiotherapy (XRT) and chemotherapy. Twenty-one patients were enrolled in the study: 16 with glioblastoma multiforme (GBM) and 5 with anaplastic astrocytoma. Twenty patients received the targeted 44-Gy boost (+/-10%) to the SCRC. Attributable toxicity was mild and limited to reversible grade 3 neutropenia or thrombocytopenia (n = 3; 14%), CNS wound infections (n = 3; 14%), and headache (n = 2; 10%). With a median follow-up of 151 weeks, median overall survival times for all patients and those with GBM are 96.6 and 90.6 weeks, respectively; 87% of GBM patients are alive at 1 year. It is feasible to consistently achieve a 44-Gy boost dose to the SCRC margin with patient-specific dosing of (131)I-81C6. Our study regimen ((131)I-81C6 + XRT + temozolomide) was well tolerated and had encouraging survival. To determine if selection of good-prognosis patients affects outcome associated with this approach, the U.S. Food and Drug Administration has approved a trial randomizing newly diagnosed GBM patients to either our study regimen or standard XRT plus temozolomide.

Eplerenone attenuates myocardial fibrosis in the angiotensin II-induced hypertensive mouse: involvement of tenascin-C induced by aldosterone-mediated inflammation.

Tenascin-C is an extracellular matrix glycoprotein that is supposed to be a profibrotic molecule in various fibrogenic processes. To elucidate its significance for myocardial fibrosis in the hypertensive heart, we used a mouse model with infusion of angiotensin II and examined results by histology, immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Angiotensin II treatment elevated blood pressure and expression of tenascin-C by interstitial fibroblasts in perivascular fibrotic lesions, and angiotensin II infusion caused accumulation of macrophages. It also upregulated expression of collagen Ialpha2; IIIalpha1; and proinflammatory/profibrotic mediators including transforming growth factor beta (TGFbeta), platelet-derived growth factor alpha (PDGF-A), PDGF-B, and PDGF-receptor alpha, but not IL-1beta and PDGF-receptor beta, in the myocardium. Treatment with an aldosterone receptor antagonist, eplerenone, significantly attenuated angiotensin II-induced fibrosis, expression of tenascin-C, and inflammatory changes without affecting the blood pressure level. In vitro, neither eplerenone nor aldosterone exerted any influence on tenascin-C expression of cardiac fibroblasts, whereas angiotensin II, TGF-beta1, and PDGF significantly upregulated expression of tenascin-C. These results suggest that, in the angiotensin II-induced hypertensive mouse heart: (1) tenascin-C may be involved in the progression of cardiac fibrosis and (2) aldosterone may elicit inflammatory reactions in myocardium, which might, in turn, induce tenascin-C synthesis of fibroblasts through at least 2 pathways mediated by TGF-beta and PDGF-A-B/PDGF-receptor alpha.

Inhibition of human embryonic palatal mesenchymal cell cycle by secalonic acid D: a probable mechanism of its cleft palate induction.

OBJECTIVE: To assess the mechanism(s) of cleft palate induction by secalonic acid D (SAD) in human embryonic palatal mesenchymal (HEPM) cells and compare them with those evaluated in the murine embryonic palate. DESIGN: Effect of SAD on HEPM cell proliferation was studied by obtaining dose response curves for cell numbers, uptake of 3H-thymidine and the expression of proliferating cell nuclear antigen (PCNA). Effects of SAD on cell cycle were assessed by flowcytometry. Cell-labeling with 3H-glucosamine and immunoblot analysis were conducted to study SAD effects on the synthesis of glycosaminogycans (GAG) and the expression of fibronectin and tenascin, respectively. RESULTS: SAD induced a concentration-dependent decrease in HEPM cell number and 3H-thymidine uptake beginning at 0.1 microg of SAD/ml. Expression of PCNA and progression of cell cycle from G1 to S phase were inhibited following SAD exposure. Cell viability was significantly reduced only at 7.5 microg/ml of SAD or higher indicating that the reduction in cell numbers by SAD at lower concentrations is likely due to reduced proliferation and at higher concentrations due to both reduced proliferation and cell death. Synthesis of extra cellular matrix components (GAGs, fibronectin or tenascin) by HEPM cells, however, was not inhibited by SAD. CONCLUSION: The results of these studies confirmed those of our previous studies with mice and the MEPM cells that SAD may induce cleft palate by reducing numbers of palatal mesenchymal cells by inhibition of their proliferation thereby leading to a reduction in the size of the developing palate shelves.

Regulation of tenascin expression in cultured rat dental pulp cells.

Tenascin (TN) is a glycoprotein of extracellular matrix abundantly present in embryonic mesenchymal tissues. Transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and retinoic acid (RA) are important regulators of dentinogenesis. Dental pulp cells have the capacity to differentiate into odontoblast-like cells. In this study, we investigated the effects of growth factors on TN expression and adhesive function using rat clonal dental pulp cells, RPC-C2A. Analyses of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting revealed that RPC-C2A cells expressed TN molecules and that TGF-beta1, HGF, and RA increased expression of TN at the mRNA and protein level, while bFGF and EGF showed a weak effect. An adhesion assay revealed that treatment with TGF-beta1, HGF, and RA induced a marked reduction of cell attachment to fibronectin (FN)-coated surfaces, whereas there was no change with bFGF and EGF. Functional blocking of growth factor-stimulated TN protein by pretreating cells with anti-TN antibodies restored cell attachment to control levels. These findings suggest that TGF-beta1, HGF, and RA may regulate pulpal cell adhesion to FN-coated surfaces and that this effect is mediated by TN.

Transforming growth factor-beta-induced transition of fibroblasts: a model for myofibroblast procurement in tissue valve engineering.

BACKGROUND AND AIM OF THE STUDY: The selection of a suitable cell type for scaffold seeding, its isolation and adequate expansion in vitro remains a major challenge in tissue valve engineering. The study aim was to establish a model for efficient procurement of myofibroblasts for in-vitro seeding using fibroblasts as progenitor cells. METHODS: Dermal and arterial mesenchymal cells from human (hDMC1.1 and hAMC1.1) and sheep (sDMC1.1 and sAMC1.1) were isolated by sequential culture. Cell isolates were characterized by stringent criteria based on morphology, immunocytochemistry using antibodies to vimentin, cytokeratin, prolyl 4-hydroxylase, smooth muscle alpha-actin (alpha-SMA) and smooth muscle myosin, and by Western blotting for alpha-SMA and N-cadherin. Cultures with less than 10-20% alpha-SMA-positive cells were considered to be fibroblastic. Cells were subsequently transdifferentiated with the cytokine transforming growth factor-beta1 (TGF-beta1) during five days, and then evaluated morphotypically, by immunocytochemistry, and by Western blotting. The metabolic and functional properties of TGF-beta1-treated and untreated colonies were compared by measuring the expression of extracellular proteins (collagen type 1 and tenascin-C) and by a collagen matrix contraction assay. RESULTS: TGF-beta1 successfully transformed both human and sheep fibroblasts to metabolically active and functional myofibroblasts based on stringent criteria for myofibroblast characterization. Alpha-SMA positivity of 100% was obtained in all cases (hDMC1.1, hAMC1.1, sDMC1.1, and sAMC1.1) after transformation compared to less than 50% in the non-transformed state (hAMC1.1, 17%; hDMC1.1, 10%; sAMC1.1, 43%; and sDMC1.1, 30%). This observation was further supported by increased contractility and an up-regulation of extracellular protein production in transdifferentiated cells. CONCLUSION: Untreated arterial cell isolates were, at best, less than 50% alpha-SMA-positive. By allowing procurement of high densities of myofibroblasts in a relatively short time, the model was seen to be a potentially useful tool in tissue valve engineering, at least in investigations using autologous cells in the sheep model.

Involvement of matrix metalloproteinases and tenascin-C in elastin calcification.

Elastin degeneration and calcification occur in many cardiovascular diseases, including medial arterial elastocalcinosis, atherosclerosis, and bioprosthetic heart valve mineralization. In the present study, we tested the hypothesis that the onset and progression of elastin-oriented calcification is associated with matrix remodeling and elastin degradation events. We studied whether aluminum ions inhibit elastin calcification by reducing elastin degradation and altering remodeling events. Subdermal implantation of pure elastin in juvenile rats resulted in a time-dependent calcification of elastin, reaching high levels 21 days after implantation. In situ hybridization showed that elastin calcification was associated with an up-regulation of matrix metalloproteinase (MMP) mRNA expression, specifically MMP-9 and MMP-2. Gelatin zymography demonstrated increased MMP-9 and MMP-2 enzyme activities in early stages of elastin calcification. Calcified elastin displayed a time-dependent pattern of tenascin-C (TN-C) and alkaline phosphatase (AP) expression. Pretreatment of pure elastin with aluminum ions prior to implantation resulted in complete inhibition of elastin calcification. Aluminum ion binding to elastin was found to protect elastin against MMP-mediated degradation in vitro. Noncalcified, explanted aluminum-pretreated elastin exhibited reduced activities of MMPs. TN-C expression in elastin implants exhibited a time-dependent pattern that was also affected by pretreatment of elastin with aluminum ions. In conclusion, elastin calcification is accompanied by matrix remodeling events, and the efficacy of aluminum pretreatment in inhibiting elastin calcification may be related in part to its effects on elastin remodeling.

A comparison of the efficacy of long-acting beta 2-agonists: eformoterol via Turbohaler and salmeterol via pressurized metered dose inhaler or Accuhaler, in mild to moderate asthmatics. Force Research Group.

Four hundred and sixty nine patients were randomized to receive either 12 micrograms bd of eformoterol (Oxis, Astra Pharmaceuticals Ltd., Kings Langley, U.K.) delivered via Turbohaler or 50 micrograms bd salmeterol (Serevent, Glaxo-Wellcome Ltd., Uxbridge, U.K.) via either the Accuhaler (Glaxo-Wellcome Ltd.) or pressurized metered dose inhaler (pMDI, Glaxo-Wellcome Ltd.) for 8 weeks. This was followed by a 4-week cross-over period when patients who had received salmeterol in the previous 8 weeks were given eformoterol and patients who had received eformoterol were given either salmeterol via the Accuhaler or pMDI to assess patient device and treatment preference. For the primary efficacy variable, the increase in peak expiratory flow (PEF) rate from run-in to 8 weeks, similar significant improvements were seen in all three treatment groups. Eformoterol Turbohaler (FT) achieved a greater increase in morning PEF than salmeterol Accuhaler (SA) from randomisation to 4 weeks; the increase shown in the eformoterol Turbohaler group was 28.9 1 min-1 compared to 19.91 min-1 for the salmeterol Accuhaler group. The addition of eformoterol Turbohaler 12 micrograms bd, to patients' existing asthma therapy was found to have a significantly more beneficial effect on the severity of patients' daytime asthma symptoms than had salmeterol Accuhaler 50 micrograms bd (P = 0.014). Eformoterol Turbohaler reduced the severity of daytime asthma symptoms by 42% after only 4 weeks of treatment. The patients in the eformoterol Turbohaler treated group experienced a higher percentage of days when they were symptom-free and did not use their short-acting bronchodilator to relieve symptoms (32.8, 24.1 and 28.0% in the FT, SA and SM groups, respectively). At 8 weeks there were no significant differences in any of these variables between the three groups. Patients in all the treatment groups gained an additional 1-1.5 nights undisturbed by asthma per week. The changes in sleep disturbance were not significantly different between the three treatment groups. In addition to the therapeutic benefits provided by eformoterol Turbohaler the device (Turbohaler) was the significant preference of patients given both Turbohaler and pMDI (P = 0.0168) and was also considered to be significantly more convenient to carry around than the Accuhaler (P < 0.0001). No other differences were found between the three devices. The results of this study demonstrate that the addition of a long-acting beta 2-agonist is an effective tool for achieving the goals of asthma treatment. Eformoterol via the Turbohaler is at least as effective as salmeterol via either the Accuhaler or the pMDI in achieving these goals.

Tenascin-C degradation in chronic wounds is dependent on serine proteinase activity.

Degradation of extracellular matrix (ECM) components by proteinases is part of the physiological remodelling process during normal wound healing. Excessive degradation of the ECM, however, is likely to create an environment that can no longer support keratinocyte migration and is thought to play a role in the impaired healing of chronic ulcers. Tenascin-C is an ECM component that is markedly upregulated in acute and chronic wounds. Here we report on our investigations into the degradation of tenascin-C in chronic venous leg ulcers. We found proteolytic fragments of tenascin-C in leg ulcer exudate. We also detected fragments of fibronectin in the wound fluid and in addition observed breakdown of fibronectin by wound fluid in vitro, as has previously been reported by others. Wound fluid of four out of six chronic leg ulcers degraded purified human tenascin-C in vitro, and degradation of tenascin-C correlated with high levels of functionally active leucocyte elastase and metalloproteinases in the wound fluid. To identify which proteinases were involved in tenascin-C degradation, we tested the effect of specific proteinase inhibitors. The addition of EDTA or E64 did not protect tenascin-C from degradation, suggesting that neither metalloproteinases nor cysteine proteinases are responsible for cleavage. Tenascin-C breakdown was inhibited by PMSF and SKALP/elafin, and we therefore conclude that leucocyte elastase and possibly other serine proteinases are the tenascin-C-degrading enzymes in ulcer exudate. Taking into account the possible effects of tenascin-C and tenascin-C fragments on cell behaviour, we hypothesize that degradation of tenascin-C could affect the healing process in chronic venous ulcers.

Tenascin-C is not a useful marker for disease activity in psoriasis.

Tenascin-C is an extracellular matrix glycoprotein that is markedly upregulated in the dermis of psoriatic skin. In this study, we have addressed the question whether the presence of tenascin-C in the lesion or in serum is a marker for disease activity. Immunohistochemical staining of tenascin-C before and after treatment with different topical and systemic medication showed that tenascin-C remained abundant after clinical remission of lesions, indicating that downregulation of tenascin-C to normal values is a slow process. By using a sensitive enzyme-linked immunosorbent assay to measure levels of serum tenascin-C in psoriatic patients and unaffected individuals, we found that tenascin-C levels in most patients were within the normal range. Moreover, tenascin-C values did not correlate with disease activity. We conclude that tenascin-C is not useful as a marker for disease activity in psoriasis.

Ozone alters the expression of tenascin-C in cultured primate nasal epithelial cells.

Tenascin-C is an extracellular matrix component which is transiently expressed in association with epithelial cell detachment, proliferation, and migration. This molecule has been identified in respiratory tissue, but little is known about the cellular source of tenascin-C or the factors that regulate its production. Since air pollutants are known to disrupt epithelial integrity, we investigated the regulation of tenascin-C in response to 0.3 ppm ozone in differentiated primate nasal epithelial cells in culture at an air-medium interface. The expression of tenascin-C was upregulated in response to ozone, as determined by Northern blot analysis, Western blotting, and immunofluorescent staining. In contrast, there was no change in the mRNA levels for versican, biglycan, perlecan, or collagen type I. Reduced cellular attachment to the substrate was evident in ozone-treated cultures in association with tenascin-C deposition at the interfaces between cells and basal surfaces. The presence of tenascin-C on denuded areas of the matrix suggests that tenascin-C may have been instrumental in the loss of patches of cells. The modulation of tenascin-C synthesis and distribution may play a significant role in the response of respiratory epithelial cells to ozone exposure.

Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid.

Tenascin and fibronectin are extracellular matrix glycoproteins expressed during morphogenesis and tissue repair. In the present study bronchial biopsies were studied by the morphometric method of immunocytochemistry to reveal the distribution of different tenascin and fibronectin isoforms as well as the presence of inflammatory cells in the airway mucosa of patients with chronic asthma (n = 32) and those with seasonal birch-pollen-sensitive asthma out of season (n = 17), both in comparison with healthy control subjects (n = 12). The results showed an increase in tenascin immunoreactivity in the bronchial subepithelial reticular basement membrane layer in patients with chronic asthma (p < 0.0001) and in those with seasonal asthma (p < 0.01) compared with control subjects. The tenascin immunoreactivity, appearing as an intense wide subepithelial band in asthma, was seen only occasionally in the basement membrane of control specimens. Instead, a diffuse immunoreaction against both total fibronectin and locally produced extradomain A fibronectin was similarly visible in the airway mucosa of both patients and control subjects. Despite the significant increase in the airway mucosa of eosinophils and lymphocytes in patients with chronic asthma (p < 0.0001 and p < 0.0001, respectively) and of eosinophils in patients with seasonal asthma (p < 0.001), there was no correlation between the number of these cell types and level of tenascin expression. In patients with birch-pollen-sensitive asthma during the birch-pollen season, inhaled corticosteroid treatment, budesonide 400 micrograms twice daily, decreased tenascin immunoreactivity, in comparison with effects of placebo (p = 0.01). Our results suggest that the higher amount of tenascin reflects disease activity in asthma and may be an indicator of a remodeling process rather than of injury itself.

Regulation of gene expression in cultured embryonic mouse mandibular mesenchyme by serotonin antagonists.

During murine embryogenesis, uptake sites for the neurotransmitter serotonin (5-HT) are transiently expressed in craniofacial epithelial structures. Based on malformations produced in cultured mouse embryos exposed to uptake inhibitors or receptor ligands, we have proposed that 5-HT acts as a dose-dependent morphogenetic signal during critical periods of craniofacial development. Several 5-HT receptor subtypes are co-distributed with tenascin and the calcium binding protein S-100 beta in developing craniofacial mesenchyme. Since these molecules are thought to be important for craniofacial development, their regulation by 5-HT could mediate some of its morphogenetic actions. Mandibular mesenchyme cells, from E12 mouse embryos (plug day = E1), grown in micromass cultures were used as an in vitro model to investigate whether 5-HT regulates expression of these molecules. Immunocytochemistry revealed expression of S-100 beta, tenascin, cartilage proteoglycan core protein (a component of the cartilage matrix) and a variety of 5-HT receptors in these cultures. To block the actions of 5-HT (from serum in the culture medium), cultures were exposed to one of these selective 5-HT receptor antagonists and effects on expression were investigated using quantitative immunobinding and in situ hybridization assays. These antagonists differentially regulated expression of cartilage core protein, S-100 beta and tenascin. Antagonism of 5-HT3 receptors by Zofran or 5-HT1A receptors by NAN-190 reduced the amount of core protein, whereas antagonism of 5-HT2A-C receptors by mianserin had no significant effect. All three antagonists stimulated levels of tenascin mRNA and protein. Expression of S-100 beta mRNA and protein was inhibited by Zofran and stimulated by mianserin, whereas NAN-190 had no significant effect. The differential effects of antagonists suggest that in vivo, 5-HT could: (1) promote expression of cartilage core protein by activation of 5-HT3 or 5-HT1A receptors, (2) inhibit production of tenascin by activation of multiple receptors, (3) promote or inhibit synthesis of S-100 beta by activation of 5-HT3 or 5-HT2 receptors, respectively. These actions may be important components of the morphogenetic functions of 5-HT during craniofacial development.

Expression of tenascin-C by human endometrial adenocarcinoma and stroma cells: heterogeneity of splice variants and induction by TGF-beta.

Localization of tenascin-C in vivo and cell culture experiments in vitro have provided evidence for stromal production of tenascin-C in malignant tumors of a variety of organs. Here we raised the question of whether the mesenchymal stroma in the case of endometrial adenocarcinoma is the unique source of tenascin-C. Therefore, the expression of tenascin-C mRNA by human endometrial adenocarcinoma cells and endometrial stroma cells was investigated. Several preparations of endometrial stroma cells produced tenascin-C mRNA. Using a serum-free defined cell culture medium, production of tenascin-C mRNA could be increased by adding either serum or 20 ng TGF-beta/mL to the cell culture medium. Reverse transcriptase polymerase chain reaction analysis revealed that five out of six endometrial adenocarcinoma cell lines produced tenascin-C mRNA. Northern blot experiments and ribonuclease protection assays provided evidence that the number of copies of tenascin-C mRNA was small. Analysis of expressed splice variants by reverse transcriptase polymerase chain reaction analysis revealed the abundance of one major splice variant that lacked all potential alternatively spliced fibronectin type-III-like repeats. Regarding larger splice variants, all fragment sizes that could theoretically originate from seven alternatively spliced fibronectin type-III-like repeats were observed. Evaluating relative signal intensities, the splice variants containing a single fibronectin type-III-like repeat and the variant possessing all but one alternatively spliced repeats were most frequent. In summary, evidence is provided that tenascin-C can originate from both tissue compartments of the human endometrium stroma and (tumor) epithelium. Splice variant analysis revealed a high number of splice variants and a relative high proportion of variants that have so far been regarded as minor constituents of expressed tenascin-C.