TGF-β1 Induction of p21WAF1/Cip1 Requires Smad-Independent Protein Kinase C Signaling Pathway

김용기 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.6

Although it has been demonstrated that p21WAF1/Cip1 could be induced by transforming growth factor-β1 (TGF-β1) in a Smad-dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood. In this study, we investigated a possible role of protein kinase C (PKC) signaling pathway in TGF-β1 induction of p21WAF1/Cip1 in human keratinocytes HaCaT cells. Our data show that PKC is required for TGF-β1 induction of p21WAF1/Cip1, as evidenced by the fact that specific inhibition of PKC leads to a decrease in p21WAF1/Cip1 protein and mRNA expression induced by TGF-β1. And this notion is further supported by the observation that activation of p21WAF1/Cip1 promoter activity is dramatically attenuated by treatment with PKC inhibitor. However, PKC signaling pathway is not associated with TGF-β1 activation of Smad signaling pathway, because inhibition of PKC signaling pathway does not affect nuclear translocation of Smads induced by TGF-β1. Taken together, our data suggest that PKC signaling pathway is required for p21WAF1/Cip1 expression by TGF-β1, which is independent of Smad signaling pathway.

EpH4 세포에서 TGF-β에 의한 세포사멸시 Smad 단백질에 의존한 Gadd45b 유전자의 발현 변화

조희준(Hee Jun Cho),유지윤(Jiyun Yoo) 한국생명과학회 2008 생명과학회지 Vol.18 No.4

Transforming growth factor-β (TGF-β)에 의해 유도되는 세포사멸 과정은 정상 조직에서 손상 받은 조직이나 비정상적인 조직을 제거하는데 중요한 역할을 담당한다. Gadd45b는 p38 kinase를 활성화시킴으로 TGF-β에 의해 유도되는 세포사멸 과정을 매개한다고 알려져 있다. 본 연구에서는 TGF-β에 의해 세포사멸이 일어나는 EpH4 세포에서 Gadd45b 유전자의 발현이 TGF-β에 의해 촉진됨을 보여주었다. 어떠한 기작으로 TGF-β에 의해 Gadd45b 유전자의 발현이 촉진되는지 알아보기 위해 Gadd45b 유전자의 5'-flanking region을 cloning하였으며, EpH4 세포에서 TGF-β에 의해 그 promoter activity가 증가함을 확인하였다. 여러 가지 deletion mutants를 제조하여 promoter activity를 조사한 결과 전사 개시점으로부터 220 bp upstream 부위에 promoter activity에 필수적인 sequence가 존재함을 확인하였다. 또한 TGF-β에 의한 Gadd45b 유전자의 promoter activity에 Smad2, Smad3, 그리고 Smad4가 중요한 기능을 담당함도 확인하였다. 마지막으로 ras 유전자가 도입되어 TGF-β에 의한 세포사멸이 억제되어있는 EpRas 세포에서 TGF-β에 의한 Gadd45b 유전자의 발현을 확인한 결과 EpRas 세포에서 TGF-β에 의한 Gadd45b 유전자의 발현이 억제됨을 확인하였다. 이러한 결과는 Gadd45b 유전자가 EpH4 세포에서 TGF-β에 의한 세포사멸을 유도하는데 중요한 기능을 담당할 가능성이 높음을 의미하는 것이다. Transforming growth factor-β (TGF-β)-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-β-induced apoptosis by the activation of p38 kinase. In this report, we show that Gadd45b is an immediate-early response gene for TGF-β during apoptosis in EpH4 cells. To elucidate the molecular mechanism of TGF-β-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-β. 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-β through the action of Smad2, Smad3, and Smad4. Finally, we show that the expression of Gadd45b gene by TGF-β is suppressed in EpRas cells in which TGF-β could not induce apoptosis, suggesting that Gadd45b may be a crucial target for TGF-β-induced apoptosis in EpH4 cells.

Soft Agar Assay를 이용한 배양골조직과 골세포의 Transforming Growth Factor-β유리에 관한 연구

백정화,김관식,정동균 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.2

To study the effect of PTH, osteotropic hormone, on the TGF-β activity in conditioned medium prepared from bone explants, fetal rat ulnae and radii were removed at 19-day of gestation and organ cultured for 24 hours. Then media were changed with fresh BGJb media for control group or PTH-supplemented media (50 or 200 ng/㎖) for experimental group respectively and conditioned media were prepared by 2-day culture of explants. To study the cellular orgin of matrix-associatied TGF-β and the effect of PTH on the TGF-β activity in conditioned media prepared from bone cells, five bone cell populations were prepared from fetal rat calvaria by sequential enzyme digestion. After primary culture, each bone cell population was collected and resuspended as Ⅰ-Ⅱ, Ⅲ and Ⅳ-Ⅴ groups. After 24 hours, media were changed with fresh minimum essential medium(MEM) or 600 ng/㎖ PTH-supplemented media and conditioned media were prepared by 2-day culture. For activation of latent TGF-β activity in bone cell-conditioned media, media were acidified and then neutralized. TGF-β activities of conditioned media were measured by anchorage-independent growth of NRK fibroblasts using modified Todaro soft agar assay method(1987) and the number of colonies ≥ 50 ㎛ was counted. The observed results were as follows. 1. TGF-β, without 2 ng/㎖ EGF, did not induce colony formation in soft agar suspensions. 2. With 2 ng/㎖ EGF, TGF-β induced NRK cells to form large colonies in soft agar suspensions and colony numbers increased proportionally to TGF-β concentrations from 50 pg/㎖ to 1600 pg/㎖. 3. When ulnae and radii were incubated with 50 ng/㎖ PTH, TGF-β activity in conditioned media was not significantly different from control conditioned media. In contrast, TGF-β activity in conditioned media prepared from culture with supplementation of 200 ng/㎖ PTH, was significantly increased(p < 0.01). 4. TGF-β activity was detectable in all bone cell populations and the amount was not significantly different between cell populations. 5. When bone cell populations were incubated with 600 ng/㎖ PTH. TGF-β activity in conditioned media from each population was not significantly different from control conditioned media. These results suggest that all cell populations isolated from calvaria synthesize TGF-β and PTH has no apparent effects on the TGF-β production of bone cell populations. In addition, increased TGF-β activity in bone explant conditioned media by 200 ng/㎖ PTH appears to be due to the enhanced release of TGF-β from bone matrix secondary to bone resorptive process.

Cinvited Review : Targeting the Transforming Growth Factor-β Signaling in Cancer Therapy

( Yhun Yhong Sheen ),( Min Jin Kim ),( Sang A Park ),( So Yeon Park ),( Jeong Seok Nam ) 한국응용약물학회 2013 Biomolecules & Therapeutics(구 응용약물학회지) Vol.21 No.5

TGF-β pathway is being extensively evaluated as a potential therapeutic target. The transforming growth factor-β (TGF-β) signaling pathway has the dual role in both tumor suppression and tumor promotion. To design cancer therapeutics successfully, it is important to understand TGF-β related functional contexts. This review discusses the molecular mechanism of the TGF-β pathway and describes the different ways of tumor suppression and promotion by TGF-β. In the last part of the review, the data on targeting TGF-β pathway for cancer treatment is assessed. The TGF-β inhibitors in pre-clinical studies, and Phase I and II clinical trials are updated.

Differential Expression of TGF-β Isoforms in Human Kerationocytes by Narrow Band UVB

( Moon Chul Jung ),( Min Kyung Shin ),( Kyung Kook Hong ),( Ki Heon Jeong ),( Nack In Kim ) 대한피부과학회 2008 Annals of Dermatology Vol.20 No.3

Background: Transforming growth factor-β (TGF-β), a multifunctional growth factor, has three isoforms: TGF-β1, TGF-β2, and TGF-β3. Different isoforms of TGF-β are associated with different proliferation and differentiation states of the epidermis. Narrow band ultraviolet B (NBUVB) emits a concentrated UVB source of 311 nm. NBUVB 1,000 mJ/cm2 induces apoptosis in approximately 50% of keratinocytes. Objective: The purpose of this study was to evaluate whether irradiation with NBUVB would alter the expression and production of TGF-β1, 2, and 3. Methods: We measured TGF-β1, 2, and 3 mRNA and TGF-β1 and 2 protein levels at 800, 1,000, and 1,200 mJ/cm2 for 24 hours and 48 hours. Results: TGF-β1 mRNA levels were increased at both 24 hr and 48 hr, TGF-β2 mRNA levels were decreased at both 24 hr and 48 hr, and TGF-β3 mRNA levels were increased at 24 hr and similar to control at 48 hr. TGF-β1 protein levels were increased at 48 hr but decreased at 24 hr. TGF-β2 protein levels were decreased at both 24 hr and 48 hr. Conclusion: The results suggest a possible role for TGF-β1 after NBUVB irradiation and opposing roles for TGF-β1 and TGF-β2 isoforms in NBUVB irradiation. (Ann Dermatol(Seoul) 20(3) 113~119, 2008)

Expression of angiogenin, TGF-β, VEGF, APEX and TNF-α in oral squamous cell carcinoma

Ho-Sun Lee,Kyoung-Won Kim,Wun-Jae Kim 대한구강악안면외과학회 2006 대한구강악안면외과학회지 Vol.32 No.1

Purpose: The purpose of this study was to verify that the expressions of angiogenin, transforming growth factor-beta(TGF-β), vascular endothelial growth factor(VEGF), human apurinic/apyrimidinic endonuclease(APEX) and tumor necrosis factoralpha(TNF-α) were associated with the tumorigenesis of the oral squamous cell carcinoma(OSCC). Materials and Methods: Fifty-one samples of OSCC and fifteen normal oral mucosae were obtained to analyze the expression levels of above five factors. mRNA expressions were quantified by the quantitative competitive PCR(QC-PCR) method. After 2% agarose gel electrophoresis stained with ethidium bromide, the concentration of mRNA was calculated by a digital image analysis system. The expression levels of angiogenin, TGF-β, VEGF, APEX and TNF-αwere compared by unpaired Student’s ttests between cancer and normal tissues. We analyzed statistically to find the cut-off values that would be useful as diagnostic markers, and the linear regression analysis between every two factors of these five factors by SAS system. Results: All of these five factors (angiogenin: P<0.0037, TGF-β: P<0.0001, VEGF: P<0.0102, APEX: P<0.0023, TNF-α: P<0.0074) were significantly correlated with OSCC. In the analysis to find the cut-off values for the diagnosis, we could not find any value that had a reasonable sensitivity and specificity. In the linear regression analysis, there were correlations between angiogenin and TNF-α, TGF-βand VEGF, TGF-βand APEX, TGF-βand TNF-α, VEGF and APEX, VEGF and TNF-α, APEX and TNF-α. Conclusion: Our results suggest that not only angiogenin, TGF-β, VEGF, APEX and TNF-αare significantly associated with the tumorigenesis, but also the close relationship between these factors might enhance the tumorigenesis of OSCC. We can not find clinical availability for diagnosis.

Synergistic Effect of Combined Growth Factors in Porcine Intervertebral Disc Degeneration

Cho, Hongsik,Lee, Sangmin,Park, Sang-Hyug,Huang, Jinsong,Hasty, Karen A.,Kim, Song-Ja Informa Healthcare 2013 Connective tissue research Vol.54 No.3

<P>Although intervertebral disc (IVD) degeneration is one of most common causes of morbidity, its etiology remains unclear. In healthy discs, the rates of synthesis and breakdown of the extracelluar matrix (ECM) are in equilibrium because of intricate regulation by growth factors and catabolic cytokines. Important among these physiologic growth factors are transforming growth factor-β (TGF-β1) and bone morphogenetic protein-2 (BMP-2). Disc degeneration is thought to be associated with a loss of this homeostasis between proteoglycan (PG) synthesis and cytokine-induced degradation leading to up-regulation of matrix metalloproteinases (MMP) families and down-regulation of extracelluar matrix production. Several strategies using biological agents have been attempted to manage IVD degeneration, improving the function and anabolic capabilities of IVD cells and inhibiting matrix degradation. The purpose of this study is to compare the effects of the anabolic cytokines BMP-2 and TGF-β1 with those of the catabolic cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) on porcine annulus fibrosus (AF). The results of this study show that the application of pro-inflammatory cytokines like tumor necrosis factor-α and interleukin-1β to normal annulus fibrosus cells leads to a significant increase in tissue levels of the degradative protease MMP-1. Treatment with a combination of minimum doses of both BMP-2 and TGF-β1 caused a greater decrease in MMP-1 and increase in aggrecan than either cytokine alone, suggesting a synergistic effect of the combined cytokines.</P>

Increased Expression of the TGF-β Isoform and Changed Contents of Collagen in Tendon of Cerebral Palsy Patients

Sung Taek Jung(정성택),Hyoung Yeon Seo(서형연),Jae Joon Lee(이재준),Myung Sun Kim(김명선),Yang Kyung Kim(김양경),Gye Jin Kim(김계진) 대한정형외과학회 2004 대한정형외과학회지 Vol.39 No.5

목적: 뇌성마비 환자의 아킬레스건 내 TGF-β isoform의 발현 정도와 콜라겐 구성을 알고자 하였다. 대상 및 방법: 뇌성마비로 인한 경직성 첨족마비 환자 중 수술적 연장술을 시행하였던 15명 환자의 아킬레스건 중앙부를 약 2×2×5 ㎣ 크기로 채취한 후, 면역조직화학 염색과 RT-PCR을 이용하여 TGF-β와 콜라겐 구성을 조사하였다. 결과: 아킬레스건 중앙부의 구성은 섬유모세포와 콜라겐 섬유 등의 기질로 구성되어 있었다. 면역조직화학 염색상 TGF-β1과 TGF-β2는 15예 중 각각 2예, 4예에서만 발현되었으며, TGF-β3의 경우는 7예에서 약하게 발현되었다. 경직성이 심한 비보행성 마비를 보인 7예 중 6예에서 TGF-β3의 양성 소견을 보였다. 콜라겐 염색상 경직성이 심한 비보행성 마비 환자에서는 상대적으로 미성숙형태인 제3형의 콜라겐이 증가됨을 알 수 있었다. 면역조직화학 염색 및 RT-PCR상 TGF-β3가 중등도 이상 발현된 7예에서 제3형 콜라겐이 증가되어 있었다. 결론: 뇌성마비로 인한 지속적인 근 구축은 건내 콜라겐 성분의 변화를 초래하고 이 과정중 TGF- 가 관여할 것으로 사료된다. Purpose: This study measured the expression level of the transforming growth factor-β(TGF-β) isoform expression and the collagen composition within the Achilles tendon from cerebral palsy (CP) patients. Materials and Methods: The Achilles tendons were obtained from 15 CP patients with spastic equinovarus. The presence of the TGF-β isoform and the composition of the collagen were examined histologically, performing by immunohistochemical staining (IHS) and determining the mRNA expression level using a reverse transcriptase polymerase chain reaction (RT-PCR). Results: IHS revealed the presence of TGF-β1 and TGF-β2 expression in 2/15 cases and 4/15 cases respectively, and weak TGF-β3 expression in 7/15 cases. The TGF-β1 and TGF-β2 expression levels were uniform in all 15 cases according to RT-PCR, while TGF-β3 expression was observed in 8 out of 15 cases. IHS and RT-PCR showed strong TGF-β3 expression in 6/7 non-ambulatory patients. An immature form of collagen, type Ⅲ collagen, was observed more abundantly in the non-ambulatory patients. Conclusion: These results suggest that contracture in CP may induce changes in the type of collagen via growth factors such as TGF-β.

초석잠 뿌리 분획물의 항산화 및 항염증 효과와 smad 신호 전달에 미치는 효과

이정우,최명원,임선영 한국생명과학회 2024 생명과학회지 Vol.34 No.4

본 연구는 초석잠 뿌리 분획물의 항산화 및 항염증 효과를 규명하고 transforming growth factor (TGF) β 자극에 의한 smad 신호전달에 미치는 효과에 대해 연구하였다. 초석잠 뿌리 분획물들 중 n-BuOH 분획물이 16.67 mg/g 으로 가장 높은 플라보노이드 함량을 나타내었으며, 다음으로 n-Hexane, water 및 85% aq. MeOH 분획물 순이었다. 초석잠 뿌리의 지방산 조성은 n-6 지방산(54.27%) > n-3 지방산(21.18%) > 포화지방산(19.70%) > n-9 지방산(3.60%) 순으로 나타났으며, 지방산들 중 n-6계 지방산인 linoleic acid (18:2n-6)가 54.27%로 나타내었고 n-3계 지방산인 linolenic acid (18:3n-3)는 20.82%의 함량을 나타내었다. DPPH 및 ABTS 라디칼 소거능을 확인한 결과, 초석잠 뿌리 분획물들 중 n-BuOH 분획물은 0.5 mg/mL 의 농도에서 각각 85.4% 및 90.2%로 라디칼 소거능을 나타내었다. 85% aq. MeOH 분획물은 0.5 mg/mL 의 농도에서 각각 58.7% 및 90.8%로 DPPH 및 ABTS 라디칼 소거능을 보였다. 항염증 효능 중 NO 생성 저해 효과는 모든 분획물들이 0.5, 1 및 2 mg/mL의 농도에서 control 과 유의적 차이를 보이면서 NO 생성을 저해하였다(p<0.05). TGFβ signaling의 smad3 의 인산화 발현량을 확인한 결과 85% aq. MeOH 및 water 분획물들을 처리한 군에서 control과 비교했을 때 각각 88% 및 77% 수준으로 smad3의 인산화가 저해되는 것을 확인하였다. 이상의 결과들로부터 플라보노이드 함량이 높은 n-BuOH 분획물의 항산화 활성이 높았고 항염증 및 TGFβ signaling의 smad3 의 인산화 저해 활성은 85% aq. MeOH 및 water 분획물들에서 높은 활성이 나타났다. We investigated to analyze total flavonoid content and fatty acid composition of Stachys sieboldii Miq root. In order to determine antioxidant and anti-inflammatory effects of fractions from S. sieboldii Miq. root, we conducted 1.1-Diphenyl-2-picryhydrazyl (DPPH) and 2,2′-Azino-bis (3-ethylbenothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS) assays for antioxidant and measured nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 cells. In addition, we examined an inhibitory effect of fractions from S. sieboldii Miq. root on smad signaling induced by transforming growth factor (TGF) β. Among the fractions, n-butanol (n-BuOH) fraction showed the highest flavonoid content (16.67 mg/g), followed by n-Hexane, water and 85% aqueous methanol (85% aq. MeOH) fractions. The fatty acid composition of S. sieboldii Miq. root was in the following order : n-6 fatty acids (54.3%) > n-3 fatty acids (21.2%) > saturated fatty acids (19.7%) > n-9 fatty acids (3.6%). As a result of the antioxidant efficacy, the DPPH and ABTS assays showed that n-BuOH fraction had higher scavenging activity compared to other fractions. Inhibitory effect on NO production showed that all fractions decreased LPS-induced NO production, indicating an anti-inflammatory activity of S. sieboldii Miq. root. 85% aq. MeOH and water fractions showed a higher efficacy in inhibiting transforming growth factor (TGF) β induced smad signaling. From the results, it suggests that food processing products using S. sieboldii Miq. root will be developed as a functional food for promoting health.

Regulation of Tumor Immune Surveillance and Tumor Immune Subversion by TGF-β

Hae-Young Park,Lalage M Wakefield,신서자 대한면역학회 2009 Immune Network Vol.9 No.4

Transforming growth factor-β (TGF-β) is a highly pleiotropic cytokine playing pivotal roles in immune regulation. TGF-β facilitates tumor cell survival and metastasis by targeting multiple cellular components. Focusing on its immunosuppressive functions, TGF-β antagonists have been employed for cancer treatment to enhance tumor immunity. TGF-β antagonists exert anti-tumor effects through #1 activating effector cells such as NK cells and cytotoxic CD8+ T cells (CTLs), #2 inhibiting regulatory/suppressor cell populations, #3 making tumor cells visible to immune cells, #4 inhibiting the production of tumor growth factors. This review focuses on the effect of TGF-β on T cells, which are differentiated into effector T cells or newly identified tumor- supporting T cells.