• The Journal of Korean Medicine
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• v.24 no.2
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• pp.1-11
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• 2003

Objectives : The aim of this study was to characterize the effect of Injinchunggan-tang on $TGF-{\beta}1-induced$ hepatic fibrosis. Methods : mRNA and protein expression levels of $TGF-{\beta}1$ in Injinchunggan-tang-treated HepG2 cells were compared to untreated cells using quantitative RT-PCR and ELISA assay, respectively. mRNA expression levels of the TGF-1 pathway genes (TR-1, TR-II, Smad2, Smad3, Smad4, and PAI-1) and fibrosis-associated genes (CTGF, fibronectin, and collagen type 1) were evaluated by quantitative RT-PCR. The effect of Injinchunggan-tang on cell proliferation of T3891 human fibroblast was evaluated using [$^3H$]thymidine incorporation assay. Results : Expression of $TGF-{\beta}1$ mRNA and protein was inhibited by Injinchunggan-tang in a dose- and time-dependent manner. Whereas $TGF-{\beta}1-mediated$ induction of PAI-1 was suppressed by Injinchunggan-tang, expression of the $TGF-{\beta}1$ pathway genes such as TR-1, TR-II, Smad2, Smad3, and Smad4 was not affected by Injinchunggan-tang treatment. Injinchunggan-tang was found to inhibit $TGF-{\beta}1-induced$ cell proliferation of T3891 human fibroblast, and also abrogated $TGF-{\beta}1-mediated$ transcriptional up-regulation of CTGF, fibronectin, and collagen type I. Conclusions : This study strongly suggests that the liver cirrhosis-suppressive activity of Injinchunggan-tang may be derived at least in part from its inhibitory effect on $TGF-{\beta}1$ functions, such as blockade of $TGF-{\beta}1$ stimulation of fibroblast cell proliferation and fibrosis-related gene expression as well as expression of $TGF-{\beta}1$ itself.

• Molecules and Cells
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• v.24 no.2
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• pp.283-287
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• 2007

$TGF-{\beta}1$ induces Ig germ-line ${\alpha}$ ($GL{\alpha}$) transcription and subsequent class switching recombination (CSR) to IgA. In the present study, we investigated the roles of two E3-ubiquitin ligases, Smurfs (HECT type) and Arkadia (RING finger type) on $TGF{\beta}1$-induced IgA CSR. We found that over-expression of Smurf1 and Smurf2 decreased $TGF{\beta}1$-induced $GL{\alpha}$ promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Further, over-expression of Smurf1 and Smurf2 decreased both Smad3/4-mediated and Runx3-mediated $GL{\alpha}$ promoter activities, suggesting that the Smurfs can down-regulate the major $TGF-{\beta}1$ signaling pathway and decrease $GL{\alpha}$ gene expression. In parallel, the over-expressed Smurf1 decreased the expression of endogenous IgA CSR-predictive transcripts ($GLT_{\alpha}$, $PST_{\alpha}$, and $CT_{\alpha}$) and also $TGF{\beta}1$-induced IgA secretion. Conversely over-expression of Arkadia abolished the inhibitory effect of Smad7 on $TGF{\beta}1$-induced $GLT_{\alpha}$ expression and IgA secretion. Similar results were obtained in the presence of over-expressed Smad7 and Smurf1. These results indicate that Arkadia can amplify $TGF{\beta}1$-induced IgA CSR by degrading Smad7, which interacts with Smurf1. We conclude that Smurf and Arkadia have opposite roles in the regulation of $TGF{\beta}1$-induced IgA isotype expression.

• Journal of Acupuncture Research
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• v.31 no.1
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• pp.7-21
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• 2014

Objectives : To observe the regenerative effects of indirect moxibustion, a traditional Korean medical treatment on skeletal muscles using mouse model of skeletal muscle adiposity. Methods : Twenty seven ICR male mice were randomly assigned into Intact control(n=3), glycerol treatment together without moxibustion(n=12), and glycerol treatment together with moxibustion (n=12) groups. Mice of glycerol treatment groups were injected with 50 ${\mu}l$ DW(distilled water) containing 50 % of glycerol into the two tibialis anterior. After injection, moxibustion was applied at 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) acupoints three times per each session, every days for twelve days(total 12 treatments). Phospho-Erk1/2, Myostatin protein levels were analyzed by western blotting and immunofluo-rescence staining techniques for tissues of the tibialis anterior muscle. Smad, phospho-Smad were analyzed by immunofluorescence staining. Results : 1. Histological analysis of sections from injected TA muscles showed that glycerol induced rapidly muscle necrosis, with a maximum at day 3. 6 days and 9 days after injection, muscle was regenerating. 2. According to western blotting and immunofluorescence staining, phospho-Erk1/2 protein signals in glycerol treatment with moxibustion group were stronger compared to Intact and glycerol treatment without moxibustion group. 3. According to western blotting and immunofluorescence staining, myostatin protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 4. According to immunofluorescence staining, Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 5. According to immunofluorescence staining, phospho-Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. Conclusions : These results confirm that indirect moxibustion of 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) influences muscle regeneration in mouse models of skeletal muscle adiposity. Further discussion, and the establishment of moxibustion mechanism will prompt clinical application of moxibustion.

• BMB Reports
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• v.52 no.3
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• pp.202-207
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• 2019

Keloids are the most common pathological form of trauma healing, with features that seriously affect appearance and body function, are difficult to treat and have a high recurrence rate. Emerging evidence suggests that miRNAs are involved in a variety of pathological processes and play an important role in the process of fibrosis. In this study, we investigated the function and regulatory network of miR-152-5p in keloids. The miRNA miR-152-5p is frequently downregulated in keloid tissue and primary cells compared to normal skin tissue and fibroblasts. In addition, the downregulation of miR-152-5p is significantly associated with the proliferation, migration and apoptosis of keloid cells. Overexpression of miR-152-5p significantly inhibits the progression of fibrosis in keloids. Smad3 is a direct target of miR-152-5p, and knockdown of Smad3 also inhibits fibrosis progression, consistent with the overexpression of miR-152-5p. The interaction between miR-152-5p and Smad3 occurs through the Erk1/2 and Akt pathways and regulates collagen3 production. In summary, our study demonstrates that miR-152-5p/Smad3 regulatory pathways involved in fibrotic progression may be a potential therapeutic target of keloids.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.4
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• pp.345-359
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• 2024

Deposition of extracellular matrix (ECM) in the trabecular meshwork (TM) increases aqueous humour outflow resistance leading to elevation of intraocular pressure (IOP) in primary open-angle glaucoma, which remains the only modifiable risk factor. Resveratrol has been shown to counteract the steroid-induced increase in IOP and increase the TM expression of ECM proteolytic enzymes; however, its effects on the deposition of ECM components by TM and its associated pathways, such as TGF-β-SMAD signalling remain uncertain. This study, therefore, explored the effects of trans-resveratrol on the expression of ECM components, SMAD signalling molecules, plasminogen activator inhibitor-1 and tissue plasminogen activator in dexamethasone-treated human TM cells (HTMCs). We also studied the nature of molecular interaction of trans-resveratrol with SMAD4 domains using ensemble docking. Treatment of HTMCs with 12.5 µM trans-resveratrol downregulated the dexamethasone-induced increase in collagen, fibronectin and α-smooth muscle actin at gene and protein levels through downregulation of TGF-β1, SMAD4, and upregulation of SMAD7. Downregulation of TGF-β1 signalling by trans-resveratrol could be attributed to its effect on the transcriptional activity due to high affinity for the MH2 domain of SMAD4. These effects may contribute to resveratrol's IOP-lowering properties by reducing ECM deposition and enhancing aqueous humour outflow in the TM.

• Journal of Life Science
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• v.18 no.4
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• pp.461-466
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• 2008

Transforming growth $factor-{\beta}$ ($TGF-{\beta}$)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Gadd45b has been known to participate in $TGF-{\beta}-induced$ apoptosis by the activation of p38 kinase. In this report, we show that Gadd45b is an immediate-early response gene for $TGF-{\beta}$ during apoptosis in EpH4 cells. To elucidate the molecular mechanism of $TGF-{\beta}-induced$ Gadd45b gene expression, we cloned the 5'-flanking region of the mouse Gadd45b gene. When transfected into EpH4 cells, this 5'-flanking region conferred promoter activity and inducibility by $TGF-{\beta}$. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 220 bp upstream of the transcription initiation site. We also found that the proximal Gadd45b promoter is activated by $TGF-{\beta}$ through the action of Smad2, Smad3, and Smad4. Finally, we show that the expression of Gadd45b gene by $TGF-{\beta}$ is suppressed in EpRas cells in which $TGF-{\beta}$ could not induce apoptosis, suggesting that Gadd45b may be a crucial target for $TGF-{\beta}-induced$ apoptosis in EpH4 cells.

• Molecules and Cells
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• v.44 no.1
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• pp.38-49
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• 2021

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of the gel-forming MUC5AC protein, are significant risk factors for patients with asthma and chronic obstructive pulmonary disease (COPD). The transforming growth factor β (TGFβ) signaling pathway negatively regulates MUC5AC expression; however, the underlying molecular mechanism is not fully understood. Here, we showed that TGFβ significantly reduces the expression of MUC5AC mRNA and its protein in NCI-H292 cells, a human mucoepidermoid carcinoma cell line. This reduced MUC5AC expression was restored by a TGFβ receptor inhibitor (SB431542), but not by the inhibition of NF-κB (BAY11-7082 or Triptolide) or PI3K (LY294002) activities. TGFβ-activated Smad3 dose-dependently bound to MUC5AC promoter. Notably, TGFβ-activated Smad3 recruited HDAC2 and facilitated nuclear translocation of HDAC2, thereby inducing the deacetylation of NF-κB at K310, which is essential for a reduction in NF-κB transcriptional activity. Both TGFβ-induced nuclear translocation of Smad3/HDAC2 and deacetylation of NF-κB at K310 were suppressed by a Smad3 inhibitor (SIS3). These results suggest that the TGFβ-activated Smad3/HDAC2 complex is an essential negative regulator for MUC5AC expression and an epigenetic regulator for NF-κB acetylation. Therefore, these results collectively suggest that modulation of the TGFβ1/Smad3/HDAC2/NF-κB pathway axis can be a promising way to improve lung function as a treatment strategy for asthma and COPD.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2083-2087
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• 2014

Colorectal cancers remain to be a common cause of cancer-related death. Early-onset cases as well as those of various ethnic origins have aggressive clinical features, the basis of which requires further exploration. The aim of this work was to examine the expression patterns of $p15^{INK4b}$ and SMAD4 in colorectal carcinoma of different ethnic origins. Fifty-five sporadic colorectal carcinoma of Egyptian origin, 25 of which were early onset, and 54 cancers of Finnish origin were immunohistochemically stained with antibodies against $p15^{INK4b}$ and SMAD4 proteins. Data were compared to the methylation status of the $p15^{INK4b}$ gene promotor. $p15^{INK4b}$ was totally lost or deficient (lost in ${\geq}50%$ of tumor cell) in 47/55 (85%) tumors of Egyptian origin as compared to 6/50 (12%) tumors of Finnish origin (p=7e-15). In the Egyptian cases with $p15^{INK4b}$ loss and available $p15^{INK4b}$ promotor methylation status, 89% of cases which lost $p15^{INK4b}$ expression were associated with $p15^{INK4b}$ gene promotor hypermethylation. SMAD4 was lost or deficient in 25/54 (46%) tumors of Egyptian origin and 28/48 (58%) tumors of Finnish origin. 22/54 (41%) Egyptian tumors showed combined loss/deficiency of both $p15^{INK4b}$ and SMAD4, while $p15^{INK4b}$ was selectively lost/deficient with positive SMAD4 expression in 24/54 (44%) tumors. Loss of $p15^{INK4b}$ was associated with older age at presentation (>50 years) in the Egyptian tumors (p=0.04). These data show for the first time that $p15^{INK4b}$ loss of expression marks a subset of colorectal cancers and ethnic origin may play a role in this selection. In a substantial number of cases, the loss was independent of SMAD4 but rather associated with $p15^{INK4b}$ gene promotor hypermethylation and old age which could be related to different environmental exposures.

• BMB Reports
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• v.38 no.1
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• pp.9-16
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• 2005

Transforming Growth Factor (TGF)-$\beta$ family, including TGF-$\beta$, bone morphorgenic protein (BMP), and activn, plays an important role in essential cellular functions such as proliferation, differentiation, apoptosis, tissue remodeling, angiognesis, immune responses, and cell adhesions. TGF-$\beta$ predominantly transmits the signals through serine/threonine receptor kinases and cytoplasmic proteins called Smads. Since the discovery of TGF-$\beta$ in the early 1980s, the dysregulation of TGF-$\beta$/Smad signaling has been implicated in the pathogenesis of human diseases. Among signal transducers in TGF-$\beta$/Smad signaling, inhibitory Smads (I-Smads), Smad6 and Smad7, act as major negative regulators forming autoinhibitory feedback loops and mediate the cross-talking with other signaling pathways. Expressions of I-Smads are mainly regulated on the transcriptional levels and post-translational protein degradations and their intracellular levels are tightly controlled to maintain the homeostatic balances. However, abnormal levels of I-Smads in the pathological conditions elicit the altered TGF-$\beta$ signaling in cells, eventually causing TGF-$\beta$-related human diseases. Thus, exploring the molecular mechanisms about the regulations of I-Smads may provide the therapeutic clues for human diseases induced by the abnormal TGF-$\beta$ signaling.

• Genomics & Informatics
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• v.20 no.1
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• pp.5.1-5.10
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• 2022

Non-syndromic hearing loss (NSHL) is a common hereditary disorder. Both clinical and genetic heterogeneity has created many obstacles to understanding the causes of NSHL. The present study has attempted to ravel the genetic aetiology in NSHL progression and to screen out potential target genes using computational approaches. The reported NSHL target genes (2009-2020) have been studied by analyzing different biochemical and signaling pathways, interpretation of their functional association network, and discovery of important regulatory interactions with three previously established miRNAs in the human inner ear as well as in NSHL such as miR-183, miR-182, and miR-96. This study has identified SMAD4 and SNAI2 as the most putative target genes of NSHL. But pathogenic and deleterious non-synonymous single nucleotide polymorphisms discovered within SMAD4 is anticipated to have an impact on NSHL progression. Additionally, the identified deleterious variants in the functional domains of SMAD4 added a supportive clue for further study. Thus, the identified deleterious variant i.e., rs377767367 (G491V) in SMAD4 needs further clinical validation. The present outcomes would provide insights into the genetics of NSHL progression.