Effect of mutant β‐catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice

Background: Mutations in the Wnt signalling pathway molecule β‐catenin are associated with liver cancer. Aims: Our aim was to confirm the effects of stabilized β‐catenin on liver growth, identify whether those effects were reversible and cell autonomous or non‐cell autonomous and to model β‐catenin‐induced liver cancer in mice. Methods: Using a liver‐specific inducible promoter, we generated transgenic mice in which the expression of mutant β‐catenin can be induced or repressed within hepatocytes in mice of different ages. Results: Similar to other models, the hepatic expression of mutant β‐catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two‐fold liver enlargement and development of disease with a latency of 1–5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β‐catenin could not increase the growth of transgene‐expressing foci in either growth‐permissive or ‐restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. Conclusions: Our findings indicate that hepatocyte gene expression is directly affected by mutant β‐catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte‐specific expression of mutant β‐catenin is secondary to liver functional alteration or non‐cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene‐expressing cells progressed to carcinoma, confirming the association of mutant β‐catenin with liver cancer.     

人脐静脉内皮细胞损伤后迁移能力的变化与血管内皮钙黏蛋白和连环蛋白p120的关系

目的初步探讨人脐静脉内皮细胞(HUVEC)损伤之后迁移能力的变化与血管内皮钙黏蛋白(VE-cad)和连环蛋白p120(p120ctn)的关系。方法 DMEM培养基培养HUVEC,将HUVEC分为对照组和损伤组。Transwell实验检测HUVEC迁移能力的变化。Western blot测定p120ctn与VE-cad蛋白表达水平。免疫荧光实验检测VEcad的定位表达变化。免疫共沉淀法检测p120ctn与VE-cad的相互结合。结果 Transwell实验发现HUVEC经损伤刺激6、8 h后迁移能力最强(P0.05)。Western blot结果显示HUVEC损伤6、8 h后p120ctn及VE-cad表达水平明显上调。免疫荧光实验显示HUVEC经损伤刺激后,VE-cad的定位由细胞膜转到细胞浆。免疫共沉淀证实p120ctn可以与VE-cad相互结合。结论 HUVEC损伤刺激后迁移能力增强,其机制可能与升高的p120ctn将VEcad由细胞膜携带入细胞浆导致VE-cad膜表达缺失有关。     

β-连环蛋白在胃腺瘤和胃癌中的表达及其意义

β-连环蛋白（eatenin）是Wnt信号转导途径异常激活的关键环节，多种肿瘤的发生、发展与B．连环蛋白异常表达密切相关。目的：观察β-连环蛋白在正常胃黏膜、胃腺瘤和胃癌组织中的表达，探讨其在胃癌发生、发展中的作用。方法：取得经胃镜活检病理检查证实的78例不同胃组织标本，包括24例正常胃黏膜、24例胃腺瘤性息肉（胃腺瘤，14例为低级别上皮内瘤变，10例为高级别上皮内瘤变）和30例胃腺癌组织，以免疫组化ABC法检测标本中β-连环蛋白的表达。结果：β-连环蛋白在正常胃黏膜和低级别上皮内瘤变胃腺瘤组织中为细胞膜表达，在高级别上皮内瘤变胃腺瘤和胃腺癌组织中呈细胞质／细胞核异位表达。胃腺癌组织中β-连环蛋白的异常表达率显著高于胃腺瘤组织（100％对41．7％，P〈0．05），膜表达缺失者占76．7％（23／30）。结论：β-连环蛋白异常表达与胃癌的发生、发展密切相关，可能是胃癌发生的早期事件。     

OBG‐like ATPase 1 inhibition attenuates angiotensin II‐induced hypertrophic response in human ventricular myocytes via GSK‐3beta/beta‐catenin signalling

Obg‐like ATPase 1 (OLA1) that possesses both GTP and ATP hydrolyzing activities has been shown to be involved in translational regulation of cancer cell growth and survival. Also, GSK3β signalling has been implicated in cardiac development and disease. However, the role of OLA1 in pathological cardiac hypertrophy is unknown. We sought to understand the mechanism by which OLA1 regulates GSK3β‐β‐Catenin signalling and its functional significance in angiotensin‐II (ANG II)‐induced cardiac hypertrophic response. OLA1 function and its endogenous interaction with GSK3β/β‐catenin signalling in cultured human ventricular cardiomyocytes (AC16 cells) and mouse hearts (in vivo) was evaluated with/without ANG II‐stimulated hypertrophic response. ANG II administration in mice increases myocardial OLA1 protein expression with a corresponding increase in GSK3β phosphorylation and decrease in β‐Catenin phosphorylation. Cultured cardiomyocytes treated with ANG II show endogenous interaction between OLA1 and GSK3β, nuclear accumulation of β‐Catenin and significant increase in cell size and expression of hypertrophic marker genes such as atrial natriuretic factor (ANF; NPPA) and β‐myosin heavy chain (MYH7). Intriguingly, OLA1 inhibition attenuates the above hypertrophic response in cardiomyocytes. Taken together, our data suggest that OLA1 plays a detrimental role in hypertrophic response via GSK3β/β‐catenin signalling. Translation strategies to target OLA1 might potentially limit the underlying molecular derangements leading to left ventricular dysfunction in patients with maladaptive cardiac hypertrophy.     

Wnt10b诱导再生毛囊的表达特性研究

目的 研究Wnt10b诱导再生毛囊的表达特性及诱导作用机制。 方法 HEK-293细胞内扩增并用氯化铯梯度离心纯化Wnt10b过表达腺病毒及对照腺病毒,皮内注射至C57BL/6J小鼠背部皮肤,在处理后2.5、5、7、9、14、28 d时取材,HE染色及免疫组化染色观察毛囊结构特征、信号通路表达特征及增殖特性。 结果 HE染色发现,AdWnt10b处理组从第5天开始出现新生毛囊结构,正常生长,第28天左右进入退化期。免疫组化染色发现,AdWnt10b处理组从处理后5 d开始新生毛囊具有AE15表达,随着毛囊生长而增加,至处理后28 d开始减少。在AdWnt10b处理后5 d,观察到β连环素的核表达,Lef1特异性表达于毛芽和毛母质部位,且全为核表达。在AdWnt10b处理后28 d,Lef1表达减弱。AdWnt10b处理后2.5 d即可见Ki67表达于表皮和毛囊外根鞘。处理后2.5、7、9、14 d均在隆突区见到Ki67的表达;从处理后7 d开始,Ki67表达于毛母质细胞。 结论 Wnt10b诱导的再生毛囊具有正常的毛囊结构,Wnt10b激活了经典Wnt信号通路,其作用的靶细胞是毛囊干细胞及其子代细胞。     

连接素的表达与舌鳞状细胞癌的转移和预后

目的：探讨连接素的表达与舌鳞状细胞癌的转移和预后的关系。方法：收集初诊舌癌标本45例，采用免疫组化SP法染色检测α－、β－连接素总阳性率均为71．1％；（2）α－、β－连接素在高分化鳞癌的阳性表达均显著高于中、低分化鳞癌；（3）有转移者α－、β－连接素阳怀表达显著低于无转移者；（4）局部肿瘤T1－2与T3－4指挥之间无统计学差异；（5）α－、β－连接素阳性表达在临床Ⅲ、Ⅳ期比Ⅰ、Ⅱ期显著降低；（6）α－、β－连接素阳性表达者5年生存率高于阴性者。结论：α－、β－连接素的表达与舌癌的病理分级，转移及预后密切相关，而与肿瘤大小无关。可用于临床指导治疗和判断预后。     

Cardiac-specific haploinsufficiency of beta-catenin attenuates cardiac hypertrophy but enhances fetal gene expression in response to aortic constriction

In addition to its role in cell adhesion, beta-catenin is an important signaling molecule in the Wnt/Wingless signaling pathway. Recent studies have indicated that beta-catenin is stabilized by hypertrophic stimuli and may regulate cardiac hypertrophic responses. To explore the role and requirement of beta-catenin in cardiac development and hypertrophy, we deleted the beta-catenin gene specifically in cardiac myocytes by crossing loxP-floxed beta-catenin mice with transgenic mice expressing a Cre recombinase under the control of the alpha-myosin heavy chain promoter. No homozygous beta-catenin-deleted mice were born alive and died before embryonic day 14.5, indicating significant and irreplaceable roles of beta-catenin in embryonic heart development. Heterozygous beta-catenin-deleted mice, however, demonstrated no structural and functional abnormality. The response of heterozygous beta-catenin-deleted mice to transverse aortic constriction, however, was significantly attenuated with decreased heart weight and heart weight/body weight ratio compared to controls with intact beta-catenin genes. Hemodynamic analysis revealed that there was no difference in cardiac function between wild-type and heterozygous beta-catenin-deleted mice. On the other hand, the expression of fetal genes, beta-myosin heavy chain, atrial and brain natriuretic peptides was significantly higher in heterozygous beta-catenin-deleted mice when compared to wild-type beta-catenin mice. These results suggest that the cytoplasmic level of beta-catenin modulates hypertrophic response and fetal gene reprogramming after pressure overload.     

Direct regulation of transforming growth factor β‐induced epithelial–mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells

Transforming growth factor β (TGFβ) causes the acquisition of epithelial–mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ‐induced phosphorylation of its C‐terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C‐terminus (PTEN4A), but not PTEN wild, inhibits TGFβ‐induced EMT. Nevertheless, whether or not the blockade of TGFβ‐induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C‐terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ‐induced EMT in lung cancer cells. The unphosphorylated PTEN C‐terminus might not directly retain the phosphatase activities and repress TGFβ‐induced EMT; the modification that keeps the PTEN C‐terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ‐induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C‐terminus might be a therapeutic target to negatively regulate TGFβ‐induced EMT in lung cancer cells.