• Biomolecules & Therapeutics
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• v.28 no.1
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• pp.98-103
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• 2020

Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 (Fbn1) gene, has vascular manifestations including aortic aneurysm, dissection, and rupture. Its vascular pathogenesis is assumed to be attributed to increased transforming growth factor β (TGFβ) signaling and blockade of excessive TGFβ signaling has been thought to prevent dissection and aneurysm formation. Here, we investigated whether galunisertib, a potent small-molecule inhibitor of TGFβ receptor I (TβRI), attenuates aneurysmal disease in a murine model of MFS (Fbn1^C1039G/+) and compared the impact of galuninsertib on the MFS-related vascular pathogenesis with that of losartan, a prophylactic agent routinely used for patients with MFS. Fbn1^C1039G/+ mice were administered galunisertib or losartan for 8 weeks, and their ascending aortas were assessed for histopathological changes and phosphorylation of Smad2 and extracellular signal-regulated kinase 1/2 (Erk1/2). Mice treated with galunisertib or losartan barely exhibited phosphorylated Smad2, suggesting that both drugs effectively blocked overactivated canonical TGFβ signaling in Fbn1^C1039G/+ mice. However, galunisertib treatment did not attenuate disrupted medial wall architecture and only partially decreased Erk1/2 phosphorylation, whereas losartan significantly inhibited MFS-associated aortopathy and markedly decreased Erk1/2 phosphorylation in Fbn1^C1039G/+ mice. These data unexpectedly revealed that galunisertib, a TβRI inhibitor, showed no benefits in aneurysmal disease in MFS mice although it completely blocked Smad2 phosphorylation. The significant losartan-induced inhibition of both aortic vascular pathogenesis and Smad2 phosphorylation implied that canonical TGFβ signaling might not prominently drive aneurysmal diseases in MFS mice.

• Journal of Periodontal and Implant Science
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• v.47 no.2
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• pp.66-76
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• 2017

Purpose: Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-${\beta}1$ (TGF-${\beta}1$). Methods: Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-${\beta}1$. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification. Results: We found that 5-aza enhanced TGF-${\beta}1$-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-${\beta}$ type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-${\beta}$ signaling. 5-aza moderately increased the expression of TGF-${\beta}$ type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-${\beta}1$. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation. Conclusions: These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-${\beta}$-induced IL11 expression in gingival fibroblasts.

• Molecular & Cellular Toxicology
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• v.5 no.3
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• pp.222-229
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• 2009

Our previous finding that pre-initiation treatment of indole-3-carbinol (I3C) represents a chemopreventive effect in dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis has prompted us to test the global expression of genes at an early stage. Rats were continuously fed 300 ppm I3C in their diet at 6 weeks of age and were injected with DMBA at 7 weeks of age, and were sacrificed at 8 weeks of age. Global gene expression analysis using oligonucleotide microarrays was conducted to detect altered genes in DMBA- or DMBA plus I3C-treated mammary glands. Altered genes were identified by fold changes of 1.2 and by t-test (P<0.05) from the log ratios of the hybridization intensity of samples between control (Group 1) and DMBA (Group 2), and from those of samples between DMBA (Group 2) and DMBA plus I3C (Group 3). From these genes, we chose altered genes that were up- or down-regulated by DMBA treatment and recovered to the control level by I3C treatment. For early stage of carcinogenesis, I3C treatment induced the recovery to normal levels of several genes including cell cycle pathway (cyclin B2, cell division cycle 2 homolog A), MAP signaling pathway (fibroblast growth factor receptor 1, platelet derived growth factor receptor, beta polypeptide), and insulin signaling (protein phosphatase 1, regulatory (inhibitor) subunit 3B and flotillin 2), which were up-regulated by DMBA treatment. In addition, I3C treatment induced the recovery to normal levels of several genes including those of MAPK signaling (transforming growth factor, beta receptor 1 and protein phosphatase 3, catalytic subunit, beta isoform), which were down-regulated by DMBA treatment. These results suggest that the targeting of these genes presents a possible approach for chemoprevention in DMBA-induced mammary carcinogenesis.

• BMB Reports
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• v.48 no.2
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• pp.115-120
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• 2015

Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development. Here, we found that TJP1 expression was increased at the RNA and protein levels in TGF-${\beta}$-stimulated lung cancer cells, A549. SB431542, a type-I TGF-${\beta}$ receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-${\beta}$ on TJP1 expression. Diphenyleneiodonium, an NADPH oxidase inhibitor, also attenuated TJP1 expression in response to TGF-${\beta}$ in lung cancer cells. When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles. Taken together, these data suggest that TGF-${\beta}$ enhances TJP1 expression, which may play a role beyond structural support in tight junctions during cancer development.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.297-306
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• 2020

The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue-PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR-14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

• Molecules and Cells
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• v.25 no.1
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• pp.105-111
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• 2008

Radiotherapy is an important treatment for many malignant tumors, but there are recent reports that radiation may increase the malignancy of cancer cells by stimulating expression of type IV collagenases. In this study, we examined changes in matrix metalloproteinase (MMP) inhibitors, such as the tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and RECK, in response to irradiation in Panc-1 pancreatic cancer cells. Irradiation increased RECK protein levels but not mRNA levels, whereas no significant changes were found in TIMP-1 and TIMP-2. The enhanced RECK protein levels were associated with an increase in MMP inhibitory activity. However, irradiation slightly but reproducibly increased the invasiveness of the Panc-1 cells. Like irradiation, treatment of Panc-1 cells with transforming growth factor $(TGF)-{\beta}1$ led to a 2-fold increase in RECK protein levels. Transient transfection with Smad3 also increased RECK protein levels, but transfection with Smad7 markedly reduced them. Stable expression of Smad7 and treatment with SB431542, an inhibitor of $TGF-{\beta}$ receptor I kinase, abolished $TGF-{\beta}1$- and radiation-mediated effects on RECK. Furthermore, irradiation increased levels of phosphorylated Smad3. We conclude that radiation post-transciptionally enhances RECK protein levels in Panc-1 cells, at least in part, via $TGF-{\beta}$ signaling, and that irradiation increases Panc-1 invasiveness via a mechanism that may not be linked to MMP-2 activity.