사람에서 Wnt 신호계의 활성화에 의한 지방세포 분화 억제와 당대사 이상과의 연관성

한호,김희수,권성준,김철근,최웅환 대한비만학회 2007 The Korean journal of obesity Vol.16 No.2

Objectives: The differentiation of preadipocyte to mature adipocyte includes activation of adipogenic gene expression and induction of insulin sensitivity. Wnt signaling, primarily mediated by Wnt10b in human, functions as a promoter of preadiocyte growth and proliferation, although it also functions as a potent inhibitor of adipogenesis. Therefore, this study was undertaken to determine the effect of Wnt signaling on the degree of adipocyte differentiation and glucose metabolism. Methods: Adipose tissue was obtained during surgery from omental fat deposits in twenty one patients who underwent elective abdominal surgery at Hanyang University Hospital. All patients were divided into NGT patients (n = 12) and patients with impaired glucose metabolism (n = 9) based on the level of the fasting plasma glucose. Tissue expression level of transcription factors for adipocyte differentiation, early and late adipocyte -specific genes, and genes for Wnt signaling and CyclinD1 were investigated in NGT patients and patients with impaired glucose metabolism through real-time quantitative PCR analysis. Results: To control for the effect of visceral adiposity on the development of impaired glucose metabolism, all patients were subdivided into low and high visceral adiposity according to the median value of V/S ratio (0.85). Among adipocyte-specific genes, expression of genes for insulin sensitivity, such as adiponectin and GLUT4 was reduced in patients with impaired glucose metabolism than NGT patients. Gene expression of Wnt10b and CyclinD1 was significantly higher in patients with impaired glucose metabolism than NGT counterparts. Conclusions: The major role of Wnt/β-catenin signaling in human adipose tissue seems to selectively inhibit genes for insulin sensitivity such as adiponectin and GLUT4, which require C/EBPα as wells as PPARγ. 목적: 비만은 인슐린 저항성을 유발하는 중요한 인자이지만, 한국인에서 제2형 당뇨병이 있는 65%의 환자들은BMI 기준을 25 kg/m2로 낮추더라도 비만하지 않다. 인슐린의 표적 세포 중 하나인 지방세포는 분화할수록 지방이축적되고 당을 흡수하는 능력이 증가한다. 인간에서 Wnt10b에 의해 매개되는 Wnt signaling의 활성화는 지방세포전구세포의 분열과 증식을 유발하지만 분화를 억제하는 것으로 알려져 있어 연구자는 인간에서 Wnt signaling의활성화 정도와 지방세포 분화 정도 그리고 당 대사와의 연관성을 알아보고자 하였다. 방법: 한양대병원 일반외과에서 선택적인 복부 수술을 시행한 21명의 환자들에서 내장 지방조직을 채취하였다. 이 환자들을 공복 혈당을 기준으로 정상군과 비정상 당 대사군 (공복혈당 장애와 제2형 당뇨병)으로 나누어 정량적중합효소반응을 통해 Wnt10b, β-catenin, Cyclin D1 (G1/S 세포주기 조절자), 지방세포 분화의 전사인자와 성숙한지방세포 특이적 유전자의 발현 정도 차이를 관찰하였다. 결과: 양 군 간에 내장 지방량의 차이를 보정하기 위해 V/S비 (visceral/subcutaneous fat ratio)의 중앙값인 0.85 를 기준으로 나누어 정상군과 비정상 당 대사군의 유전자 발현양의 차이를 비교하였다. 높은 내장 지방량을 가진환자들 중 비정상 당대사 군에서 PPARγ에 의해 그 발현이 조절되는 지방형성에 관여하는 유전자인 aP2의 발현이증가하였으나, 발현을 위해 PPARγ뿐만이 아니라 C/EBPα도 필요로 하는 인슐린 감수성을 개선하는 유전자인adiponectin과 GLUT4의 발현은 유의하게 감소하였다. 높은 내장 지방량을 가진 환자들 중 비정상 당대사 군에서Wnt10b와 Wnt signaling에 의해 활성화되는 Cyclin D1의 발현이 유의하게 증가하였다. 결론: 인간의 내장 지방세포에서 Wnt signaling의 활성화는 지방세포 형성과정에서 PPARγ에 의한 C/EBPα의활성화를 선택적으로 억제함으로써 인슐린 감수성에 관여하는 adiponectin과 GLUT4의 발현을 억제하는 것으로 생각된다.

Overexpression of Wnt5a in Primary Oral Mucosal Melanoma

You Jin Lee,Rivera Rosario,Sang Gun Ahn,Jung Hoon Yoon 대한구강악안면병리학회 2007 대한구강악안면병리학회지 Vol.31 No.2

Wnt genes encode a large fami ly of secreted cysteine-rich signaling mol ecules involved in cell growth‘ differentiation and tumorigenesis, Wnt5a, a non-transforming member of the Wnt fa mily behaves as a putative onco 一 gene in many cancers including melanomas, The aim of our study was to determine Wnt5a ex pression pattern in pnmalγ oral mucosal melanomas(Ol\αÆ) and correlate it with tumor thlckness Archival ti ssues from 18 OMM cases were subjected to immunohistochemical detection of Wnt5a by the streptavidin-biotin method , These were categorized into tumors of (4 mm(thin and intermediate thickness lesions) and )4 mm(t hi ck lesions) t hickness, Most OMM cases(17/18; 94, 4%) stained positive for Wnt5a, though heterogeneously, Seven t hi ck(7/11: 64%) and one in termediate thickness(1/7‘ 14%) ol\α‘ demonstrated strongly positive Wnt5a staining(P(O, 05), The only Wnt5a-neg ative case was a thi ck OMM without local recurrence after treatment Strong Wnt5a express ion at tumor adva ncing sites suggests a role in local tumor spread, Identification of pleomorphi c epitheli oid and spindle cells as mel anoma cell populations with the most pronounced Wnt5a staining suggests that Wnt5a overexpression inJ] uences cellular phenotype, These results taken together suggest that Wnt5a is up- regulated in OMM a nd may play a role in tumor progression, Wnt5a activity is most frequently detected in advanced OMM suggesting its function in tumor progression, Expression level of Wnt5a in OMM correlated with tumor grade and t hickness

Wnt/β-catenin 신호를 조절하는 인산화 효소

신은영(Eun-Young Shin),박창균(Edmond Changkyun Park),홍연희(Yeonhee Hong),김건화(Gun-Hwa Kim) 한국생명과학회 2013 생명과학회지 Vol.23 No.7

Wnt/β-catenin 신호는 세포의 운명 결정, 증식, 분화 등을 조절하는 척추 동물 배아 발생과 성체의 항상성 유지에 필수적인 세포신호전달경로이다. 이러한 Wnt/β-catenin의 비정상적인 조절에 의해 선천적 기형, 암, 대사질환 등을 비롯한 다양한 질병이 유발된다. 이를 바탕으로 최근 Wnt/β-catenin 신호의 조절을 통한 암을 비롯한 질병의 치료를 위한 연구가 활발히 진행되고 있다. 따라서 Wnt/β-catenin 신호를 조절하는 인자의 발굴 및 자세한 작용 기전에 대한 연구가 절실히 필요하다. 본 총설에서는 최근 새롭게 알려진 Wnt/β-catenin 신호 조절 기작에 대해 설명하고, 현재까지 알려진 Wnt/β-catenin을 조절하는 인산화 효소(kinase)의 종류와 작용 기전과 새로운 약물 타겟으로 전망을 알아 보고자 한다. The Wnt/β-catenin signaling pathway is an evolutionarily conserved signaling network that is critical for embryonic development and adult tissue maintenance. In addition, aberrant activation of Wnt/β-catenin signaling is implicated in the formation of various human diseases, including cancers. Thus, study of the underlying molecular mechanism of Wnt/β-catenin signaling regulation is important to understand and treat diseases. Inhibition of aberrant Wnt pathway activity in cancer cell lines efficiently blocks their growth, highlighting the great potential of therapeutics designed to achieve this in cancer patients. Recently, protein kinases have emerged as key regulating components of Wnt/β -catenin signaling. In this review, we provide the most recent information on Wnt/β-catenin signaling, describe protein kinases involved in Wnt/β-catenin signaling, and discuss their potential as drug targets.

Silybin Synergizes with Wnt3a in Activation of the Wnt/${\beta}$-catenin Signaling Pathway through Stabilization of Intracellular ${\beta}$-Catenin Protein

김태연,오상택,Kim, Tae-Yeoun,Oh, Sang-Taek The Korean Society for Microbiology and Biotechnol 2012 한국미생물·생명공학회지 Vol.40 No.1

Wnt/${\beta}$-catenin 신호전달체계는 세포의 분화와 증식, 기관의 발생과 조절을 담당하는 중요한 세포내 신호전달체계이다. 발생과정에서 Wnt/${\beta}$-catenin 신호전달체계의 작용이 지방세포로의 분화를 억제하고 조골세포와 신경세포로의 분화는 촉진한다는 많은 연구들이 보고되어 있으며, 현재 Wnt/${\beta}$-catenin 신호전달체계의 조절을 통한 여러 질병의 치료와 예방에 대한 관심이 대두되고 있다. 본 연구에서는 세포를 기반으로 한 초고속 저분자 스크리닝 시스템을 이용하여 Wnt의 상승제인 silybin을 발굴하였다. silybin은 Wnt가 존재 않을 경우에는 ${\beta}$-catenin 단백질의 수준에 영향을 미치지 않지만 Wnt가 존재할 경우, mRNA 발현양의 변화 없이 세포질내의 ${\beta}$-catenin 단백질의 수준을 증가시킨다. 또한 silybin에 의해 증가된 ${\beta}$-catenin으로 인해 지방세포분화에 중요한 전사인자라고 알려진 PPAR-${\gamma}$와 C/EBP-${\alpha}$의 발현을 억제한다. 따라서 이 연구에서는 silybin이 세포질내 ${\beta}$-catenin 단백질의 수준을 증가시킴으로써 Wnt/${\beta}$-catenin 신호전달체계를 활성한다는 사실을 제시하였다. The Wnt/${\beta}$-catenin signaling pathway regulates diverse developmental processes and adult tissue homeostasis. Inappropriate regulation of this pathway has been associated with human diseases, such as cancers, osteoporosis, and Alzheimer's disease. Using a cell-based chemical screening with natural compounds, we discovered silybin, a plant flavonoid isolated from the Silybum marianum, which activated the Wnt/${\beta}$-catenin signaling pathway in a synergy with Wnt3a-conditioned medium (Wnt3a-CM). In the presence of Wnt3a-CM, silybin up-regulated ${\beta}$-catenin response transcription (CRT) in HEK293-FL reporter cells and 3T3-L1 preadipocytes through stabilization of intracellular ${\beta}$-catenin protein. Silybin and Wnt3a-CM synergistically reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated $receptor{\gamma}$ ($PPAR{\gamma}$) and CAATT enhancer-binding protein ${\alpha}$ (C/$EBP{\alpha}$) in 3T3-L1 preadipocytes, accompanied by the activation of Wnt/${\beta}$-catenin signaling pathway. Taken together, our findings indicate that silybin is a small-molecule synergist of the Wnt/${\beta}$-catenin signaling pathway and can be used as a controllable reagent for investigating biological processes that involve the Wnt/${\beta}$-catenin signaling pathway.

Differential Wnt11 Expression Related to Wnt5a in High- and Low-grade Serous Ovarian Cancer: Implications for Migration, Adhesion and Survival

Jannesari-Ladani, Farnaz,Hossein, Ghamartaj,Izadi-Mood, Narges Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.3

Wnt is a powerful signaling pathway that plays a crucial role in cell fate determination, survival, proliferation and motility during development, in adult tissues and cancer. The aims of the present study were three fold: i) to assess Wnt11 immunoexpression and its possible relationship with Wnt5a in high- and low-grade human serous ovarian cancer (HGSC and LGSC) specimens; ii) to assess Wnt11 expression levels in Wnt5a overexpressing SKOV-3 cells; iii) to reveal the role of Wnt11 in viability, adhesion, migration and invasion of SKOV-3 cells using recombinant human Wnt11 (rhWnt11). Immunohistochemistry revealed a significant difference in Wnt11 expression between HGSC and LGSC groups (p=0.001). Moreover, a positive correlation was observed between Wnt5a and Wnt11 expression in the HGSC (r=0.713, p=0.001), but not the LGSC group. The expression of Wnt11 was decreased by 35% in Wnt5a overexpressing cells (SKOV-3/Wnt5a) compared to mock controls. Similarly Wnt11 expression levels were decreased by 47% in the presence of exogenous Wnt5a compared to untreated cells. In the presence of rhWnt11, 31% increased cell viability (p<0.001) and 21% increased cell adhesion to matrigel (p<0.01) were observed compared to control. Cell migration was increased by 1.6-fold with rhWnt11 as revealed by transwell migration assay (p<0.001). However, 45% decreased cell invasion was observed in the presence of rhWnt11 compared to control (p<0.01). Our results may suggest that differential Wnt11 immunoexpression in HGSC compared to LGSC could play important roles in serous ovarian cancer progression and may be modulated by Wnt5a expression levels.

Wnt5a attenuates the pathogenic effects of the Wnt/ β-catenin pathway in human retinal pigment epithelial cells via down-regulating β-catenin and Snail

( Joo Hyun Kim ),( Seoyoung Park ),( Hyewon Chung ),( Sangtaek Oh ) 생화학분자생물학회(구 한국생화학분자생물학회) 2015 BMB Reports Vol.48 No.9

Activation of the Wnt/ β-catenin pathway plays a pathogenic role in age-related macular degeneration (AMD) and is thus a potential target for the development of therapeutics for this disease. Here, we demonstrated that Wnt5a antagonized β-catenin response transcription (CRT) induced with Wnt3a by promoting β-catenin phosphorylation at Ser33/Ser37/Thr41 and its subsequent degradation in human retinal pigment epithelial (RPE) cells. Wnt5a decreased the levels of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and nuclear factor-κB (NF-κB), which was up-regulated by Wnt3a. Furthermore, Wnt5a increased E-cadherin expression and decreased cell migration by down-regulating Snail expression, thereby abrogating the Wnt3a-induced epithelial-mesenchymal transition (EMT) in human RPE cells. Our findings suggest that Wnt5a suppresses the pathogenic effects of canonical Wnt signaling in human RPE cells by promoting β-catenin phosphorylation and degradation. Therefore, Wnt5a has significant therapeutic potential for the treatment of AMD. [BMB Reports 2015; 48(9): 525-530]

Wnt7b is Upregulated in Macrophages during Thymic Regeneration and Negatively Regulated by RANKL

김종갑,김성민,김봉선,김재봉,윤식,배수경,Kim, Jong-Gab,Kim, Sung-Min,Kim, Bong-Seon,Kim, Jae-Bong,Yoon, Sik,Bae, Soo-Kyung Korean Society of Life Science 2007 생명과학회지 Vol.17 No.7

성체흰쥐의 경우 항암제인 싸이클로포스파마이드 (CY)처리로 퇴축된 가슴샘은 2주 후에 정상조직으로 재생된다. 가슴샘 발생과정에서 이미 알려진 Wnt신호전달의 중요성과는 달리 성체의 가슴샘 재생과정에서 그 역할에 관해서는 알려진 바 전혀 없다. 본 연구의 목적은 발생중인 가슴샘 상피세포에서 발현이 증가된다고 이미 알려져 있는 Wnt7b가 성체의 가슴샘재생과정에서 어떤 발현 양상을 보이는지를 조사하는 것이다. Wnt7b는 가슴샘의 급성 퇴축 이후 3일째 되는 시기에 mRNA와 단백질의 양이 급격히 증가 하였으며, 이중 면역 염색 형광법을 통해 큰포식 세포와 위치적 분포가 일치함을 확인하였다. 또한, Wnt7b유전자의 발현 조절 기전을 밝히기 위해 Wnt7b의 Reporter Vector를 제작하여 Luciferase assay를 이용하여 상위의 신호를 분석하였고, 그 결과 Wnt7b는 RANKL에 의해 그 발현이 감소된다는 사실을 처음으로 밝혔다. 따라서, 본 연구 결과들을 통해 Wnt 7b는 가슴샘의 급성 퇴축 초기 과정에서 나타나는 손상된 세포를 처리하는 큰포식 세포의 기능 조절에 관여할 것으로 생각된다. Thymus can regenerate to its normal mass within 14 days after acute involution induced by cyclophosphamide (CY) in adult rat. Despite the established role of Wnt pathways in the process of thymus development, they have not yet been associated with the regeneration of adult thymus. The purpose of this study was to investigate whether Wnt7b, which is expressed in developing thymic epithelial cells rather than in thymocytes, is modulated during thymic regeneration in adult rat. Here, we show that Wnt7b expression was up-regulated in the regenerating thymus. Cells immunolabeled for the Wnt7b were identified as macrophages. Furthermore, Wnt7b gene expression was decreased by the treatment of receptor activator of NF-kappaB ligand (RANKL). Taken together, our results demonstrate that Wnt7b gene expression was increased in macrophages during thymic regeneration and negatively regulated by RANKL.

Effect of Wnt3a on Keratinocytes Utilizing <i>in Vitro</i> and Bioinformatics Analysis

Nam, Ju-Suk,Chakraborty, Chiranjib,Sharma, Ashish Ranjan,Her, Young,Bae, Kee-Jeong,Sharma, Garima,Doss, George Priya,Lee, Sang-Soo,Hong, Myung-Sun,Song, Dong-Keun Molecular Diversity Preservation International (MD 2014 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.15 No.4

<P>Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. <I>In silico</I> phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers.</P>

각막윤부 줄기세포의 줄기세포성 유지 및 증식에 대한 Wnt 단백질의 효과

진현진,주천기,Hyun-Jin Jin,BS,Choun-Ki Joo,MD,PhD 대한안과학회 2009 대한안과학회지 Vol.50 No.4

Purpose: To evaluate the effects of the Wnt protein on proliferation and stemness maintenance of cultured corneal limbal stem cells. Methods: We examined the expression of Wnt proteins by Western blot analysis. We then evaluated the effects of Wnt on cell proliferation by colony forming efficiency. β-catenin activation using Wnt proteins was examined by immunocytochemistry. We also examined the effects of Wnt on proliferation and stemness maintenance by reverse transcriptase polymerase chain reaction of p63 and connexin43. Results: Wnt has a different effect on corneal epithelial stem cells. Colony forming efficiency was also significantly higher in treated Wnt2 and Wnt4 cells compared with controls. The Wnt2 and Wnt4 treated cells showed nuclear accumulation of β -catenin. In addition, the limbal stem cell marker p63 was strongly expressed in Wnt2, Wnt4 Wnt5a, and Wnt5b. Connexin43 mRNA was also strongly expressed in Wnt5a, Wnt5b and Wnt7b cells. Conclusions: We suggest that Wnt2 and Wnt4 could lead to more effective proliferation and stemness maintenance for human corneal epithelial stem cells. J Korean Ophthalmol Soc 2009;50(4):588-593

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model

Yoon, Hyung Ho,Lee, Hyang Ju,Min, Joongkee,Kim, Jeong Hoon,Park, Jin Hoon,Kim, Ji Hyun,Kim, Seong Who,Lee, Heuiran,Jeon, Sang Ryong The Korean Neurosurgical Society 2021 Journal of Korean neurosurgical society Vol.64 No.5

Objective : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3-asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. Methods : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×10⁵ cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. Results : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. Conclusion : Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.