Transforming Growth Factor-β1 Gene Research Articles

Gene Expression and Cytokine Release during Odontogenic Differentiation of Human Dental Pulp Stem Cells Induced by 2 Endodontic Biomaterials

IntroductionMineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) have shown osteogenic/cementogenic/dentinogenic activities; however, their mechanism of action is not fully understood. We aimed to evaluate the effect of these biomaterials on odontogenic differentiation of human dental pulp stem cells (DPSCs). MethodsFlow cytometry with stem cell markers for the confirmation of stemness and homogeneity was first performed. Then isolated DPSCs were seeded on prepared discs of MTA, CEM, differentiation medium (DM), and growth medium (GM) and incubated up to 14 days. Concentrations of transforming growth factor-β1, bone morphogenetic protein (BMP)2, BMP4, and fibroblast growth factor 4 were measured at each interval using an enzyme-linked immunosorbent assay reader. Gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and the cytokines were evaluated by reverse-transcription polymerase chain reaction. To evaluate the cell morphology, scanning electron micrographs were taken; mineralization potential was evaluated using alizarin red S staining. ResultsScanning electron micrographs showed that DPSCs spread/adhered/proliferated similarly on MTA and CEM. On day 14, alizarin red S staining confirmed that mineralization occurred in all groups except GM. Expressions of dentin matrix protein 1 and dentin sialophosphoprotein genes were similar in the CEM, MTA, and DM groups; they were significantly higher compared with the GM group (P < .05). A greater amount of transforming growth factor-β1 gene was expressed in MTA compared with the other groups (P < .05). However, the expression of fibroblast growth factor 4 and BMP2 genes was significantly greater in the CEM group (P < .05). In all the tested groups, the expression of BMP4 was less than GM (P < .01); however, CEM and DM were similar but more than MTA (P < .05). Concentrations of protein product detected using an enzyme-linked immunosorbent assay reader confirmed these gene expressions. ConclusionsMTA and CEM can induce osteo-/odontogenic-like phenotype differentiation of human DPSCs; however, they stimulate different gene expressions and growth factor release.

Deletion of Cysteine-Cysteine Receptor 7 Promotes Fibrotic Injury in Experimental Post-Thrombotic Vein Wall Remodeling

Deep vein thrombosis (VT) can result in vein wall injury, which clinically manifests as post-thrombotic syndrome. Postinjury fibrosis may be modulated in part through cellular cysteine-cysteine receptor 7 (CCR7)-mediated events. We tested the hypothesis that late vein wall fibrotic remodeling is dependent on CCR7. CCR7(-/-) and C57BL/6 wild-type mice had inferior vena cava VT induced by nonstasis or stasis mechanisms. In both models, VT size was largest at day 1 and trended down by day 21, and CCR7(+) cells peaked at day 8 in wild-type mice. No significant differences in VT resolution were found in CCR7(-/-) as compared with wild type in either model. In the nonstasis VT model, vein wall changes consistent with fibrotic injury were evidenced by significant increases in collagen I, III, matrix metalloproteinase 2, and transforming growth factor-β gene expression, increases in α-smooth muscle actin and fibroblast specific protein-1 antigen, and total collagen at 8 days. Correspondingly, SM22α and fibroblast specific protein-1, but not DDR2(+) cells, were increased at 8 days. Early wild-type thrombus exposure inhibited profibrotic gene expression in CCR7(-/-) in ex vivo vein wall culture. Bone marrow chimera experiments further showed that circulating CCR7(+) leukocytes partially rescued midterm profibrotic changes in CCR7(-/-) mice. In human histological sections of chronic thrombosed femoral veins, CCR7(+) cells were present in the fibrotic areas. Post-thrombotic vein wall remodeling is impaired in CCR7(-/-) mice, with a profibrotic phenotype, is dependent on the thrombotic mechanism, and is mediated by circulating CCR7(+) cells. Unlike other postinjury fibrotic responses, CCR7(+) signaling may be important for positive vein wall remodeling after VT.

An updated meta-analysis of transforming growth factor-β1 gene: three polymorphisms with gastric cancer

To derive a more precise estimation of the relationship between TGF-β1 polymorphisms and gastric cancer (GC) risk, we conducted a meta-analysis of all available case-control studies relating the C-509 T, T869C, and G 915C polymorphisms of the TGF-β1 gene to the risk of developing GC. The effect summary odds ratio (OR) and 95% confidence intervals (CIs) were obtained. Funnel plots and Egger's test were used to estimate publication bias. Finally, 11 studies were included in the final meta-analysis. With respect to C-509 T polymorphism, it was found that significantly increased GC risk was associated with the TT genotype in the recessive genetic model in overall analysis (TT vs. CC + CT: OR = 1.23, 95% CI 1.09-1.38, P(heterogeneity) = 0.13) and in Asian population (TT vs. CC + CT: OR = 1.24, 95% CI 1.10-1.39, P(heterogeneity) = 0.18). With respect to T869C and G915C polymorphisms, no significant association with GC risk was demonstrated in overall analysis and subgroup analyses according to ethnicity for all genetic models. This meta-analysis suggested that the T allele of TGF-β1 509C/T polymorphism is probably the susceptibility factor for GC.

Genetic variants in transforming growth factor-β gene (TGFB1) affect susceptibility to schizophrenia

Immense body of evidence indicates that dysfunction of immune system is implicated in the etiology of schizophrenia. The immune theory of schizophrenia is supported by alterations in cytokine profile in the brain and peripheral blood. Given the strong genetic background of schizophrenia, it might be assumed that aberrant production of cytokines might be the consequence of genetic factors. This study aimed at investigating the association between schizophrenia susceptibility and selected functional polymorphisms in genes encoding cytokines including: interleukin-2 (IL2 −330T>G, rs2069756), interleukin-6 (IL-6 −174G>C, rs1800795), interferon-γ (IFNG +874T>A, rs2430561) as well as for the first time transforming growth factor-β1 (TGFB1 +869T>C, rs1800470 and +916G>C, rs1800471). We recruited 151 subjects with schizophrenia and 279 controls. There was a significant difference in the genotype distribution and allelic frequency of the TGFB1 +869T>C between patients with schizophrenia and healthy controls (p < 0.05). The risk of schizophrenia was more than two-fold higher in carriers of T allele (CT+TT genotypes) than individuals with CC genotype. Given documented gender differences in incidence of schizophrenia, we conducted separate analyses of male and female participants. We have shown that the association was significant in females, while in males it reached a trend toward statistical significance. To the best of our knowledge, it is the first report showing the association between TGFB1 +869T>C polymorphism and schizophrenia.

New insights in extracellular matrix remodeling and collagen turnover related pathways in cultured human tenocytes after ciprofloxacin administration.

We characterized the effect of ciprofloxacin (CPX) in cultured human tenocytes by morphological and molecular methods. Collagen type I and III mRNA and protein levels were unaffected, but lysyl hydroxylase 2b mRNA levels progressively decreased after CPX administration. MMP-1 protein levels significantly increased after 20 μg/ml CPX administration but remained unmodified at the higher dose, whilst MMP-2 activity was unchanged. Tissue inhibitor of MMP (TIMP-1) gene expression decreased after CPX treatment, whilst TIMP-2 and transforming growth factor-β1 gene expression, the cytoskeleton arrangement, and cytochrome c expression remained unmodified. Secreted Protein Acidic and Rich in Cysteine mRNA and protein levels remained almost unchanged, whilst N-cadherin mRNA levels resulted significantly down-regulated and connexin 43 gene expression tended to decrease after CPX administration. The CPX-induced decreased ability to cross-link collagen and decreased TIMP-1 levels, possibly leading to higher activity of MMPs in ECM degradation, together with the down-regulation of N-cadherin and connexin 43 are consistent with a reduced ability to maintain tissue homeostasis, possibly making the tendon more susceptible to rupture.

Autologous minced muscle grafts: a tissue engineering therapy for the volumetric loss of skeletal muscle

Volumetric muscle loss (VML) results in a large void deficient in the requisite materials for regeneration for which there is no definitive clinical standard of care. Autologous minced muscle grafts (MG), which contain the essential components for muscle regeneration, may embody an ideal tissue engineering therapy for VML. The purpose of this study was to determine if orthotopic transplantation of MG acutely after VML in the tibialis anterior muscle of male Lewis rats promotes functional tissue regeneration. Herein we report that over the first 16 wk postinjury, MG transplantation 1) promotes remarkable regeneration of innervated muscle fibers within the defect area (i.e., de novo muscle fiber regeneration); 2) reduced evidence of chronic injury in the remaining muscle mass compared with nonrepaired muscles following VML (i.e., transplantation attenuated chronically upregulated transforming growth factor-β1 gene expression and the presence of centrally located nuclei in 30% of fibers observed in nonrepaired muscles); and 3) significantly improves net torque production (i.e., ∼55% of the functional deficit in nonrepaired muscles was restored). Additionally, voluntary wheel running was shown to reduce the heightened accumulation of extracellular matrix deposition observed within the regenerated tissue of MG-repaired sedentary rats 8 wk postinjury (collagen 1% area: sedentary vs. runner, ∼41 vs. 30%), which may have been the result of an augmented inflammatory response [i.e., M1 (CCR7) and M2 (CD163) macrophage expression was significantly greater in runner than sedentary MG-repaired muscles 2 wk postinjury]. These findings support further exploration of autologous minced MGs for the treatment of VML.

Biochemical basis of vocal fold mobilization after microflap surgery in a rabbit model

To investigate phonation-related extracellular matrix (ECM) changes in the vocal fold lamina propria after microflap surgery using an in vivo rabbit phonation model. Prospective animal study. Twenty-four New Zealand White rabbits were used in this study. Quantitative polymerase chain reaction and immunohistochemistry were used to investigate alterations in vocal fold ECM proinflammatory and profibrotic gene, and protein expression from a control group of animals receiving a microflap without phonation and a separate group of animals receiving experimentally induced phonation on postmicroflap days 0, 3, and 7. IHC demonstrated the highest concentration of CD45 in vocal folds on postoperative day 0. Staining for CD45 was absent by postoperative day 7, with no differences in CD45 staining between groups. Fibronectin gene expression increased significantly on postoperative day 3 in the control and experimentally induced phonation groups, with maximal staining of fibronectin around the microflap incision on postoperative day 7. No alterations in cyclooxygenase-2, interleukin-1β, and transforming growth factor-β1 gene expression were observed between groups. Results of the present study revealed an acute inflammatory response in the vocal fold at the time of microflap (day 0) and up to 3 days post-microflap. By post-operative day 3, staining of CD45 positive cells decreased, with essentially no evidence of inflammation by post-operative day 7. With the end of the acute inflammatory response occurring around day 3, these data may provide support for mobilizing tissue after inflammation has subsided and the process of active tissue remodeling has ensued (days 3-7). N/A.

Association of interleukin-10, tumor necrosis factor-α and transforming growth factor-β gene polymorphisms with the outcome of diffuse large B-cell lymphomas

Published data indicate that common genetic variants in immune/inflammatory response genes can affect the outcome of diffuse large B-cell lymphomas (DLBCL). This study investigated the association of interleukin (IL)-10 (-3575, -1082), tumor necrosis factor (TNF)-α -308 and transforming growth factor (TGF)-β Leu10Pro gene polymorphisms with clinical characteristics and outcome of DLBCL patients treated with rituximab-CHOP therapy. Between January 2004 and December 2007, a total of 84 patients with newly diagnosed DLBCL entered into this study. Genotypes were determined with PCR-based methodology. Patients presenting with B symptoms had IL-10 -3575 TA/AA genotypes more frequently than TT genotype [odds ratio (OR) 2.89, 95 % confidence interval (CI) 1.11-7.57; p = 0.03]. Carriers of TGF-β Pro10 allele more frequently had an advanced clinical stage III/IV (OR 4.65, 95 % CI 1.33-16.19; p = 0.016) and intermediate-high/high IPI score (OR 5.37, 95 % CI 1.45-20.0; p = 0.012). In rituximab-CHOP-treated patients (n = 64), the TNF-α -308 AG/AA carriers had shorter overall (p = 0.048) and event-free survival (p = 0.07) compared to GG carriers. In multivariate analysis of prognostic factors for survival, the TNF-α AG/AA genotypes were significantly associated with inferior survival of lymphoma patients (OR 0.23, 95 % CI 0.07-0.78; p = 0.018). Our results indicate the association of IL-10 -3575 and TGF-β Leu10Pro gene variations with clinical characteristics. In patients treated with rituximab-CHOP therapy, the TNF-α -308 AG/AA genotypes showed a significantly less favorable survival than the GG genotype.

Transforming growth factor-β1 gene mutations and phenotypes in pediatric patients with Camurati-Engelmann disease

The aim of the present study was to investigate the clinical characteristics and major causative gene in pediatric patients with Camurati‑Engelmann disease (CED). Biochemical and radiographic examinations, bone scintigraphy and genetic analyses were performed in two affected males and their parents. The two patients experienced waddling gait, muscular weakness and growth developmental delay. X-ray radiography revealed typical fusiform thickening of the diaphyseal portions of the long bones. The abnormal uptake of tracer Tc-99m was visualized in the skull and both sides of the upper humeri, ulnas, radii, femurs and tibias using bone scintigraphy. Serum levels of the bone formation marker procollagen typeI N-terminal propeptide (PINP) and the bone resorption marker β‑isomerized C-terminal cross-linked telopeptide of typeI collagen (β-CTX) in the 6-year-old patient were significantly increased compared with the normal value range, while only the β-CTX levels were elevated in the 16-year-old patient. A heterozygous missense mutation p.Arg218Cys in exon4 of the transforming growth factorβ1 (TGFβ1) gene was detected in the two patients, while their parents had normal wild‑type genotypes. In conclusion, the p.Arg218Cys mutation was shown to contribute to the clinical phenotypes in two pediatric patients with CED. The results of this study suggest that abnormal bone turnover marker levels, typical radiological findings and mutations in the TGFβ1 gene are three important factors in the diagnosis of sporadic CED cases.

Genetics of cerebral amyloid angiopathy: systematic review and meta-analysis

Background and purposeCerebral amyloid angiopathy (CAA) is common in the ageing brain and is associated with dementia and lobar intracerebral haemorrhage. We systematically reviewed genetic associations with CAA to better...

Integrin control of the transforming growth factor-β pathway in glioblastoma

Transforming growth factor-β is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-β promotes invasiveness and angiogenesis, maintains cancer cell stemness and induces profound immunosuppression in the host. Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation, differentiation and motility, and may be involved in the activation of transforming growth factor-β. We report that αvβ3, αvβ5 and αvβ8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to αv, β3 or β5 neutralizing antibodies, RNA interference-mediated integrin gene silencing or pharmacological integrin inhibition using the cyclic RGD peptide EMD 121974 (cilengitide) results in reduced phosphorylation of Smad2 in most glioma cell lines, including glioma-initiating cell lines and reduced transforming growth factor-β-mediated reporter gene activity, coinciding with reduced transforming growth factor-β protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-β bioactivity due to integrin inhibition likely results from two distinct mechanisms: an early effect on activation of preformed inactive protein, and second, major effect on transforming growth factor-β gene transcription as confirmed by decreased activity of the transforming growth factor-β gene promoter and decreased transforming growth factor-β(1) and transforming growth factor-β(2) messenger RNA expression levels. In vivo, EMD 121974 (cilengitide), which is currently in late clinical development as an antiangiogenic agent in newly diagnosed glioblastoma, was a weak antagonist of pSmad2 phosphorylation. These results validate integrin inhibition as a promising strategy not only to inhibit angiogenesis, but also to block transforming growth factor-β-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastoma.

Annexin A1: A new immunohistological marker of cholangiocarcinoma

To evaluate a new immunohistological marker, annexin A1 (ANXA1), in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC). Expression of ANXA1 protein was investigated in liver tissues from patients with CCA and HCC by immunohistochemistry. Its expression on differences stages of tumor development was investigated in hamster CCA tissues induced by Opisthorchis viverrini and N-nitrosodimethylamine. Moreover, mRNA expression of ANXA1 was assessed in CCA cell lines by quantitative real-time polymerase chain reaction and silencing of ANXA1 gene expression using small interfering RNA. In human CCA tissue arrays, immunohistochemical analysis revealed that the positive expression of ANXA1 was 94.1% (64/68 cases) consisting of a high expression (66.2%, 45/68 cases) and a low expression (33.8%, 23/68 cases). However, expression of ANXA1 protein was negative in all histologic patterns for HCC (46/46 cases) and healthy individuals (6/6 cases). In hamster with opisthorchiasis-associated CCA, the expression of ANXA1 was observed in the cytoplasm of inflammatory cells, bile duct epithelia and tumor cells. Grading scores of ANXA1 expression were significantly increased with tumor progression. In addition, mRNA expression of ANXA1 significantly increased in all of the various CCA cell lines tested compared to an immortalized human cholangiocyte cell line (MMNK1). Suppressing the ANXA1 gene significantly reduced the matrix metalloproteinase (MMP) 2 and MMP9, and transforming growth factor-β genes, but increased nuclear factor-κB gene expression. ANXA1 is highly expressed in CCA, but low in HCC, suggesting it may serve as a new immunohistochemical marker of CCA. ANXA1 may play a role in opisthorchiasis-associated cholangiocarcinogenesis.

The effect of mitotane on viability, steroidogenesis and gene expression in NCI-H295R adrenocortical cells

Mitotane, also known as o,p'‑DDD or (RS)‑1‑chl-oro‑2‑[2,2‑dichloro‑1‑(4‑chlorophenyl)‑ethyl]‑benzene, is an adrenal cortex-specific cytotoxic drug used in the therapy of adrenocortical carcinoma (ACC). The drug also inhibits steroidogenesis, however, the mechanisms of its anticancer and antisteroidogenic effects remain unknown. At present, data on the impact of mitotane on cell viability and the regulation of genes encoding proteins associated with steroids synthesis in the adrenal cortex, including cortisol and dehydroepiandrosterone sulfate (DHEAS), are limited and contradictory. In the present study, the effect of 24‑h mitotane treatment on viability of the ACC cell line, NCI‑H295R, was analyzed, identifying a decrease in cell viability and an increase in caspase‑3 and ‑7 activities. Mitotane treatment also led to decreased cortisol and DHEAS concentration in the culture media. Concomitantly, mitotane resulted in decreased mRNA levels of two cytochromes P450(CYP11A1 and CYP17A1), mRNAs encoding proteins involved in the synthesis of cortisol and DHEAS. Mitotane did not affect mRNA levels of cyclin dependent kinase inhibitor 1A (encoding p21) and MYC (encoding cMyc). cMyc and p21 are key transcription factors associated with cell cycle regulation. However, mitotane inhibited expression of transforming growth factor β1 gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. PRKAR1A, a protein kinase A regulatory subunit, is involved in the activation of steroidogenesis. PRKAR1A mRNA levels were reduced following 24‑h treatment with mitotane. Results indicate that mitotane markedly inhibited expression of genes involved in steroidogenesis, secretion of cortisol and DHEAS. Reduced expression of TGFB1 cannot account fully for the effect of mitotane on CYP11A1 and CYP17A1. We hypothesized that reduced viability of NCI‑H295R cells in the presence of mitotane may be a result of apoptosis triggered by increased caspase‑3 and ‑7 activities. Since p21 and cMyc mRNA levels were stable in the presence of mitotane, the mechanism by which caspase‑3 and ‑7 are induced remains unknown.

Association between single nucleotide polymorphisms of the transforming growth factor β1 gene and the risk of severe radiation esophagitis in patients with lung cancer

PurposeWe investigated the association between single nucleotide polymorphisms (SNPs) in the transforming growth factor β1 (TGFβ1) gene and the risk of radiation-induced esophageal toxicity (RE) in patients with non-small-cell lung cancer (NSCLC). Methods and materialsNinety-seven NSCLC patients with available genomic DNA samples and mostly treated with intensity modulated radio(chemo)therapy from 2003 to 2006 were used as a test dataset and 101 NSCLC patients treated with 3-dimensional conformal radio(chemo)therapy from 1998 to 2002 were used as a validation set. We genotyped three SNPs of the TGFβ1 gene (rs1800469:C-509T, rs1800471:G915C, and rs1982073:T869C) by the polymerase chain reaction restriction fragment length polymorphism method. ResultsIn the test dataset, the CT/TT genotypes of TGFβ1 rs1800469:C-509T were associated with a statistically significant higher risk of RE grade⩾3 in univariate (P=0.026) and multivariate analysis (P=0.045) when compared with the CC genotype. These results were again observed in both univariate (P=0.045) and multivariate (P=0.023) analysis in the validation dataset. ConclusionWe found and validated that the TGFβ1 rs1800469:C-509T genotype is associated with severe RE. This response marker may be used for guiding therapy intensity in an individual patient, which would further the goal of individualized therapy.

Metformin Inhibits Advanced Glycation End Products (AGEs)-induced Renal Tubular Cell Injury by Suppressing Reactive Oxygen Species Generation via Reducing Receptor for AGEs (RAGE) Expression

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in tubulointerstitial damage in diabetic nephropathy. Recently, metformin has been shown to ameliorate tubular injury both in cell culture and diabetic animal model. However, effects of metformin on AGEs-induced tubular cell apoptosis and damage remain unknown. We examined here whether and how metformin could block the AGEs-RAGE-elicited tubular cell injury in vitro. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. Reactive oxygen species (ROS) generation was measured with dihydroethidium staining. Apoptosis was evaluated by DNA fragmentation and annexin V expression level. AGEs upregulated RAGE mRNA levels and subsequently increased ROS generation and intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and transforming growth factor-β gene expression in human renal proximal tubular cells, all of which were significantly blocked by the treatment of 0.01 and 0.1 mM metformin. Compound C, an inhibitor of AMP-activated protein kinase significantly blocked the effects of metformin on RAGE gene expression and ROS generation in AGEs-exposed tubular cells. Furthermore, metformin dose-dependently inhibited the AGEs-induced apoptotic cell death of tubular cells; 1 mM metformin completely suppressed the pro-apoptotic effects of AGEs in 2 different assay systems. Our present study suggests that metformin could inhibit the AGEs-induced apoptosis and inflammatory and fibrotic reactions in tubular cells probably by reducing ROS generation via suppression of RAGE expression through AMP-activated protein kinase activation. Metformin may protect against tubular cell injury in diabetic nephropathy by blocking the AGEs-RAGE-ROS axis.

Transforming growth factor beta 1 genotype and p16 as prognostic factors in head and neck squamous cell carcinoma

Conclusion: Transforming growth factor β1 gene (TGFβ1) genotype is a potential p16 independent prognostic factor predicting response to chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC). Objectives: Expression of p16 and epidermal growth factor receptor (EGFR) has been reported to be associated with survival in HNSCC. We have previously reported that genetic polymorphism of TGFβ1 is linked with survival in HNSCC patients who have undergone chemoradiotherapy. We evaluate here whether TGFB1 genotype can serve as a prognostic factor independent of tumor p16 and EGFR expression. Methods: Expression of p16 and EGFR was studied by immunohistochemistry in tumors from 130 HNSCC patients. Peripheral blood DNA was used to genotype 95 patients for single nucleotide polymorphism rs1800470 within the TGFβ1 gene. The minimum follow-up time was 31 months. Results: p16 overexpression was associated with an improved disease-free survival (hazard ratio (HR) = 0.39, 95% CI 0.19–0.78), whereas no evident association was observed between EGFR expression and disease-free survival (HR = 0.90, 95% CI 0.68–1.19). Among the 37 patients who had received chemoradiotherapy, TGFβ1 genotype was associated with disease-free (HR = 0.44, 95% CI 0.19–1.02) and overall survival (HR = 0.31, 95% CI 0.12–0.80) independent of tumor p16 expression.

The mRNA level of the transforming growth factor β1 gene, but not the amount of the gene product, can be considered as a potential prognostic parameter in inflammatory bowel diseases in children

PurposeTransforming growth factor β1 (TGF-β1) plays a role in cell proliferation and differentiation, and it can modulate immune response. In this work, we asked whether levels of either TGF-β1 or mRNA of the corresponding gene in plasma or tissue can be useful in diagnosing and/or monitoring of the clinical course of inflammatory bowel diseases (IBD).MethodsThe study group consisted of 104 pediatric patients with IBD: 36 with Crohn’s disease (CD) and 68 with ulcerative colitis (UC); 42 children represented the control group. TGF-β1 levels in plasma and intestinal mucosa were estimated by ELISA and immunohistochemistry (IHC), respectively. Levels of TGF-β1 mRNA were determined by reverse transcription and real-time PCR.ResultsIn patients with IBD, and in subgroups with CD and UC, no significant differences in the TGF-β1 level in plasma and tissue were found relative to the control group. These variables were not dependent on the stage of the disease, its activity or severity of endoscopic and histopathological findings. TGF-β1 mRNA levels were significantly higher in tissue samples withdrawn during the relapse of the disease than in those taken during the remission or in the control group. However, no correlation between TGF-β1 plasma levels and TGF-β1 mRNA amount in the intestinal mucosa was observed.ConclusionsThe TGF-β1 mRNA level, but not the amount of the gene product, was significantly increased in the pathologically changed tissue during the relapse of IBD. We suggest that this parameter might be considered as a potential prognostic value when assessing IBD in children.Electronic supplementary materialThe online version of this article (doi:10.1007/s00384-012-1489-4) contains supplementary material, which is available to authorized users.

No Effect of the Transforming Growth Factor β1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

To study whether the promoter polymorphism (C-509T) affects transforming growth factor β1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

PRKCB is associated with calcineurin inhibitor-induced renal dysfunction in heart transplant recipients

Single nucleotide polymorphisms (SNPs) in the transforming growth factor-β1 gene (TGFB1) have been inconsistently associated with calcineurin inhibitor (CNI)-induced renal dysfunction following cardiac transplantation. The impact of genetic variants related to the renin-angiotensin-aldosterone system (RAAS) and natriuretic peptides, which are implicated in CNI nephrotoxicity, is unknown. The primary objective of this study was to validate the association between two common variants in TGFB1 (rs1800470, rs1800471) and postcardiac transplant renal function. The secondary objective was to investigate the effect of candidate genes related to the RAAS, natriuretic peptides, and other elements involved in the intracellular signaling of these pathways. We conducted a retrospective cohort study of 158 heart transplant recipients treated with CNIs, and evaluated the association between select SNPs and the estimated glomerular filtration rate as calculated by the Modification of Diet in Renal Disease simplified formula. A total of 273 SNPs distributed in 44 genes were tested. No association was observed between TGFB1 variants and renal function. One polymorphism in the protein kinase C-β gene (PRKCB; rs11074606), which is implicated in the RAAS intracellular signaling, was significantly associated with post-transplant estimated glomerular filtration rate after adjusting for possible confounders (P=0.00049). This marker is in linkage disequilibrium with two variants located in putative regulatory regions of the gene (rs2283541, rs1013316). Our results suggest that PRKCB may be a potential predictor of CNI-induced nephrotoxicity in heart transplant recipients, and could therefore be a promising candidate to identify patients who are most susceptible to this adverse drug reaction.

Transforming Growth Factor-β1 Regulates Cdk5 Activity in Primary Sensory Neurons

In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na(+)/Ca(2+) channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca(2+) influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.