The gap junction protein Cx43 is involved in the bone-targeted metastatic behaviour of human prostate cancer cells

For decades, cancer was associated with gap-junction defects. However, more recently it appeared that the gap junction proteins (connexins) could be re-expressed and participate to cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer (PCa) are known to be connexin deficient, it was interesting to verify whether their bone-targeted metastatic behaviour could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates to the differentiation of osteoblastic cells. Thus, we investigated the effect of the increased Cx43 expression, by retroviral infection, on the metastatic behaviour of two well-characterized cell lines (PC-3 and LNCaP) representing different stages of PCa progression. It appeared that Cx43 differently behaved in those cell lines and induced different phenotypes. In LNCaP, Cx43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those Cx43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of Cx43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of Cx43 may govern the ability of PCa cells to metastasize in bones.     

In Vivo and In Vitro Expression of Connexin 43 in Human Teeth

Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of small regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.     

Cx43在造血调控及重建中的作用

目的:采用条件性基因敲除技术构建造血系统间隙连接蛋白43(Cx43)基因敲除(Cx43~(-/-))小鼠模型,并探讨Cx43在维持造血细胞自我更新及功能稳定中的作用。方法:将引进的2对转基因小鼠Cx43 loxP/loxP和Lyz-Cre/+杂交,选取F1雌性子代Cx43 loxP/-_Lyz-Cre/+与雄性Cx43 loxP/loxP合笼回配,提取所获得子代小鼠鼠尾组织基因组DNA,采用PCR方法鉴定小鼠基因型,RT-PCR方法筛选Cx43~(-/-)小鼠,同时分析小鼠不同器官中Cx43基因的表达差异;该类小鼠经5-氟尿嘧啶(5-FU;125 mg/kg)处理,在化疗前及化疗后第5、10和15天经眼球取血分析其血象变化。Cx43~(-/-)及Cx43~(+/+)小鼠予7.5 Gy(~(60)Co-γ)的致死量照射,剂量率1 Gy/min,照射后6 h分别给予事先准备就序的骨髓细胞,每只3×10~6细胞于尾静脉注入,2周后处死小鼠检测造血是否重建:分离股骨切片后,收集骨髓细胞进行细胞表型分析(选用的单抗为CD45R、Gr-1、CD4、 CD8a、TCRαβ、Mac-1、抗sIgM、TER119、Sca-1及CD117);同时进行体外造血细胞集落实验观察造血细胞的体外增殖能力。结果:本研究通过2种转基因小鼠间杂交和回交,成功获得造血系统选择性Cx43基因敲除小鼠;该类小鼠骨髓及外周血细胞无Cx43表达,参与造血的组织,如肝脏和脾脏中Cx43表达也显著下调(P0.01),而心脏和肾脏的Cx43表达则无影响,小鼠成年后外周血象分析并无明显异常,但应急代偿能力下降,经5-FU处理后,其造血功能恢复显著减缓,处理15 d后,Cx43~(+/+)小鼠造血功能已接近正常水平,而Cx43~(-/-)小鼠仍无明显的恢复迹象,血红蛋白、白细胞及血小板仍处低位,2者差别有统计学显著性(P0.01);体外集落试验也证实Cx43~(-/-)小鼠造血干/祖细胞的增殖能力下降,其CFU-GM或CFU-E集落数均明显少于Cx43~(+/+)小鼠(P0.01),但流式细胞术结果显示,Cx43~(-/-)小鼠骨髓中Lin~-/c-Kit~+/Sca-1~+细胞亚群数量与Cx43~(+/+)小鼠相比差异并无统计学显著性;Cx43~(-/-)小鼠在化疗或移植后其骨髓造血功能重建均延迟,且化疗15 d后骨髓切片及涂片均证实其骨髓中造血细胞增生程度明显降低,脂肪组织显著增多,而且T、B细胞发育也有异常。此外,其外周血中CD4~+CD8~+细胞比例比野生型小鼠增多(P0.05),但CD4~+T细胞显著减少(P0.01),尤其是TCRαβ亚群细胞减少最为明显(P0.01)。同样,Cx43~(-/-)小鼠外周血中CD45R~+sIgM~-细胞亚群比例与野生型小鼠相比显著减少(P0.01)。结论:骨髓中Cx43基因表达在造血干/祖细胞发育(尤其是应急状态时)具重要作用,敲除Cx43基因后造血干/祖细胞增殖减缓,造血及免疫重建功能受损。     

人鼻咽不同部位粘膜上皮PCNA表达的比较研究

目的 探讨鼻咽部肿瘤发生位置差异性的机理。方法 应用免疫组织化学与图像分析技术对人鼻咽粘膜上皮增殖细胞核抗原（ＰＣＮＡ）的表达状况进行了比较研究。结果 鼻咽上皮的ＰＣＮＡ标记指数以及ＰＣＮＡ阳性细胞的分布状况均随发育时期及其所处鼻咽部位置的不同而变化，成人鼻咽的顶壁和侧壁粘膜上皮具有相对较高的ＰＣＮＡ标记指数，与人鼻咽癌的易感部位正好形成对应关系。结论 鼻咽部不同部位的粘膜上皮在上皮细胞群体增殖活     

不同妊娠时期人胎盘绒毛细胞连接蛋白基因表达的研究

目的 探索妊娠不同时期人胎盘绒毛细胞连接蛋白基因表达的规律及其与细胞分化的关系。方法 采用多种细胞连接蛋白基因ｃＤＮＡ探针,通过Ｎｏｒｔｈｅｒｎ印迹法,研究了不同妊娠时期胎盘绒毛细胞内Ｃｘ基因的表达状态。结果 １．Ｃｘ基因表达有器官特异性,绒毛细胞中Ｃｘ４３、Ｃｘ３２及Ｃｘ４０表达,而Ｃｘ３１．１、Ｃｘ３３、Ｃｘ３７、Ｃｘ４６不表达;２．在不同发育阶段的胎盘阶段的胎盘绒毛中,Ｃｘ４３、Ｃｘ３２、Ｃ     

同种异体骨髓间充质干细胞移植上调急性心肌梗死大鼠Cx43的表达

目的研究同种异体骨髓间充质干细胞(MSCs)移植心肌梗死(MI)大鼠后缝隙连接蛋白43(Cx43)在不同时期的动态变化。方法建立大鼠MI模型。将同种异体MSCs用5-氮胞苷诱导成心肌样细胞并行荧光标记,经二次开胸注射入MI大鼠梗死区和梗死边缘区。各亚组分别于移植后4、8和12周在荧光显微镜下跟踪MSCs移植情况。同时用免疫组化分析Cx43表达与缝隙连接(GJ)分布。结果MSCs体外诱导可分化为自发搏动的心肌样细胞,表达心肌特异性肌钙蛋白T(cTnT)和形成肌丝结构。MSCs移植后可长期存活并在4、8和12周,并有效上调缺血区Cx43的表达,改善GJ分布紊乱状态。在梗死区Cx43无特殊改变。结论MSCs具有分化为心肌样细胞的可塑性,移植后上调MI后缺血区Cx43表达,改善GJ分布紊乱。     

新生儿心肌连接蛋白43、45的表达

目的　研究新生儿心肌不同部位连接蛋白 4 3、4 5 (Cx4 3、Cx4 5 )表达的差异。方法　应用SP免疫组化方法 ,分析和比较 6例新生儿心不同腔室心肌细胞Cx4 3、Cx4 5的表达。结果　 1 新生儿心肌Cx4 3的蛋白表达在心四个腔均有表达 ,呈斑点状遍布于整个心房肌和心室肌的细胞质内和细胞膜表面 ,少数位于闰盘处 ;Cx4 3、主要在心室肌表达 ,心房较少 ;2 Cx4 3与Cx4 5的分布类似 ,但远少于Cx4 3。结论　Cx4 3主要分布于新生儿心肌细胞质内和细胞表面。心室表达多于心房 ,Cx4 5和Cx4 3分布相似 ,但不如Cx4 3丰富。     

Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties

HeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltage-clamp technique. Different types of cell pairs were established and their gap junction properties determined, i.e. the dependence of the instantaneous and steady-state conductances (g_j,inst, g_j,ss) on the transjunctional voltage (V_j) and the kinetics of inactivation of the gap junction current (I_j). Pairs of singly transfected cells showed homogeneous behaviour at both V_j polarities. Homotypic Cx43-Cx43 and Cx45-Cx45 cell pairs yielded distinct symmetrical functions g_j,inst=f(V_j) and g_j,ss=f(V_j). Heterotypic Cx43-Cx45 preparations exhibited asymmetric functions g_j,inst=f(V_j) and g_j,ss=f(V_j) suggesting that connexons Cx43 and Cx45 gate with positive and negative V_j, respectively. Preparations containing a singly (Cx43 or Cx45) or doubly (Cx43/45) transfected cell showed quasi-homogeneous behaviour at one V_j polarity and heterogeneous behaviour at the other polarity. The former yielded Boltzmann parameters intermediate between those of Cx43-Cx43, Cx45-Cx45 and Cx43-Cx45 preparations; the latter could not be explained by homotypic and heterotypic combinations of homomeric connexons. Each pair of doubly transfected cells (Cx43/Cx45) yielded unique functions g_j,inst=f(V_j) and g_j,ss=f(V_j). This can not be explained by combinations of homomeric connexons. We conclude that Cx43 and Cx45 form homomeric-homotypic, homomeric-heterotypic channels as well as heteromeric-homotypic and heteromeric-heterotypic channels. This has implications for the impulse propagation in specific areas of the heart.     

缝隙连接蛋白43和黄体生成素受体在小鼠卵巢中的表达

目的 观察小鼠卵泡发育过程中缝隙连接蛋白43（Cx43）和黄体生成素受体（LHR）在小鼠卵巢中的表达以及黄体生成素(LH)对卵巢Cx43表达的影响,为这两种分子与卵泡发育的关系提供实验依据。 方法 选择出生后1d、4d、7d、2周、3周、4周、6周、8周龄雌性C57小鼠共8组,每组10只,取卵巢。HE染色观察卵巢形态、卵泡发育并测量颗粒细胞体积分数。免疫组织化学和Western blotting观察卵巢组织中Cx43及LHR的表达,Western blotting检测不同浓度黄体生成素（LH）孵育后对卵巢Cx43表达的影响。 结果 HE染色显示,出生1d小鼠卵巢中见原始卵泡,出生7d卵巢内出现初级卵泡,2周时出现窦前卵泡和少量窦卵泡,3周、4周时,出现较多大的窦卵泡,6～8周时出现黄体。卵泡颗粒细胞体积分数在2周后的卵巢中明显增加。Cx43表达在各阶段卵泡的颗粒细胞胞膜上,LHR表达在卵泡膜细胞、颗粒细胞及卵母细胞胞质中。Cx43和LHR出生后2周卵巢中的表达开始明显增加,并随卵泡发育表达逐渐增强,在3周、4周、6周和8周维持相对较高水平。LH体外干预可增加卵巢组织Cx43的表达。 结论 Cx43和LHR在卵泡发育中发挥重要作用,LH可能通过LHR促进卵巢卵泡颗粒细胞Cx43的表达。     

Developmental regulation of connexin 43 expression in fetal mouse testicular cells

Multiple connexins have been identified in testicular cells. Several lines of evidences indicate that, among them, connexin 43 (Cx43) may be unique for control of gonad development and spermatogenesis. To date, however, it is not known whether Cx43 is expressed in the fetal testis and what possible types of cellular interactions mediated by this connexin are critical to male fertility. In the present work, expression of Cx43 was investigated at various developmental ages in cryosections from mouse testis by using specific antibodies against Cx43. In serial or double-labeled sections, Cx43 localization was compared with immunocytochemical distribution of steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GCNA1), which are specific markers, respectively, of interstitial Leydig, Sertoli, and germinal cells. Sections were analyzed by fluorescence microscopy. We found that Cx43 immunofluorescence (IF) was uniformly distributed in the undifferentiated gonad at 11.5 days post coitus (dpc) and in cells of the mesonephric tubules. In the undifferentiated gonad, Cx43 was localized between primordial germ cells and somatic cells. At 12.5 dpc, when the gonad has undergone sexual differentiation, in the interstitium Cx43 was localized in Leydig cells and in the seminiferous cord it was localized between adjacent Sertoli cells. In Leydig and Sertoli cells, Cx43 labeling increased at 14.5, 16.5, and 18.5 dpc. From day 12.5 up to 18.5 dpc, Cx43 was also localized in cell borders between germinal and Sertoli cells. In conclusion, this study demonstrates that from the earliest stages of gonadal development, Cx43 is expressed in the principal cell types that participate in the control of male fertility. It also shows that Cx43 expression in Leydig and Sertoli cells increase during fetal life. Finally, it provides evidence that, throughout embryonic life, Cx43 forms gap junctions between Sertoli and germinal cells.     

成人与新生儿心脏连接蛋白43表达差异

目的　探讨成人与新生儿心脏连接蛋白 4 3(Cx4 3)表达差异。　方法　应用SP免疫组织化学和图像分析方法 ,观测成人与新生儿心脏Cx4 3的蛋白表达。　结果　 1 新生儿心脏Cx4 3在心房和心室均呈斑点状遍布于心肌细胞侧面连接处和细胞质内 ,闰盘处极少。 2 成人Cx4 3表达在心房肌非均质分布于细胞侧面连接处和端闰盘处 ;心室肌典型地排列在闰盘处。 3 图像分析表明 ,心肌细胞Cx4 3分布密度 ,新生儿心房 <心室 ,成人心房 >心室。成人心房、心室均低于新生儿。　结论　新生儿Cx4 3主要分布于心肌细胞侧连接处 ,成人心房和心室存在差异。Cx4 3分布密度新生儿心房 <心室 ,成人心房 >心室 ;成人心脏低于新生儿。提示 ,Cx4 3有增龄变化。     

抑制小鼠HL-1心肌细胞桥粒斑蛋白基因表达对缝隙连接蛋白43结构和功能的影响

 目的：采用基因沉默技术抑制小鼠HL-1心肌细胞桥粒斑蛋白（DSP）基因表达以明确DSP与缝隙连接蛋白43(Cx43)的结构和功能关系。方法：用基因沉默技术抑制DSP基因的表达,Western blotting和流式细胞术检测HL-1细胞DSP和Cx43蛋白的表达,用双免疫荧光方法检测DSP与Cx43蛋白的表达与定位情况,并用划痕标记染料示踪技术检测细胞缝隙连接通讯状况。结果：与空白组和对照组相比,siRNA-DSP组的DSP和Cx43蛋白表达量降低（P＜0.05）。免疫荧光检测发现空白组和对照组DSP与Cx43蛋白存在共定位情况,而siRNA-DSP组DSP和Cx43蛋白共定位遭到破坏,Cx43蛋白出现再分布,在细胞内检测到Cx43蛋白,并且划痕标记染料示踪技术检测发现siRNA-DSP组Lucifer yellow通过HL-1细胞缝隙连接的传输功能降低。结论：DSP表达抑制不仅使Cx43出现再分布,而且影响缝隙连接传导功能。     

In vivo and in vitro expression of connexin 43 in human teeth

Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of mall regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.     

miR-301a-3p对大鼠星形胶质细胞Cx43转录后的靶向调控作用

目的:研究大鼠星形胶质细胞中微小RNA-301a-3p(miR-301a-3p)对缝隙连接蛋白43(Cx43)表达的靶向调控作用及其作用位点。方法:合成miR-301a-3p agomir和miR-301a-3p antagomir,转染至星形胶质细胞,Western blot检测各组细胞中Cx43蛋白的表达情况;构建重组载体wt-pEZX-MT05-Cx43和mut-pEZX-MT05-Cx43,采用双萤光素酶报告基因实验验证miR-301a-3p的靶基因;构建表达载体pcDNA3.1-Cx43,通过回复实验分析miR-301a-3p对细胞凋亡的影响。结果:将miR-301a-3p agomir转染到星形胶质细胞后,Western blot检测显示,与对照组相比,Cx43蛋白表达显著降低(P<0.05)。双萤光素酶报告基因实验结果表明,miR-301a-3p能够与Cx43的3′-UTR结合,对其表达产生负调控;将不含Cx43 3′-UTR的重组载体pcDNA3.1-Cx43转染星形胶质细胞后,能够回复miR-301a-3p对Cx43蛋白表达的负调控作用,引起细胞凋亡。结论:Cx43是miR-301a-3p的一个靶基因,miR-301a-3p通过作用于Cx43 mRNA的3′-UTR而抑制其在大鼠星形胶质细胞中的表达。     

Alterations in Cx43 and OB-cadherin affect breast cancer cell metastatic potential

Emerging evidence suggests that gap junctional intercellular communication (GJIC) and expression of connexins (Cx) contribute to the metastatic potential of breast cancer cells. To more directly address this, an aggressive bone metastasis breast cancer cell line, MDA-MET (MET), was stably transfected with human Cx43 cDNA (MET/Cx43⁺). Focusing on clone 28 of MET/Cx43⁺, we demonstrated that GJIC, Cx43 protein and Cx43 mRNA were significantly increased in MET/Cx43⁺ cells relative to MET, the plasmid control for the Cx43 transfectants (MET/HY) and a metastatic breast cancer cell that is less metastatic to bone than MET, MDA-MB-231. Cx26 mRNA was also increased in MET/Cx43⁺ clone 28 cells while mRNA for Cx32, Cx37, Cx40 and Cx45 were not detected in any of the breast cancer cell lines examined. MET/Cx43⁺ clone 28 invasiveness was decreased by 33% relative to MET/HY, while their ability to migrate was unchanged. The ability of MET/Cx43⁺ clone 28 cells to adhere to hFOB and HUV-EC-C cells was decreased approximately 30% and 70%, respectively, relative to MET and MET/HY. E-cadherin and N-cadherin proteins were not detected in MET, MDA-MB-231, MET/Cx43⁺ clone 28 and MET/HY cells. However, OB-cadherin protein levels were decreased approximately 43% in MET/Cx43⁺ clone 28 relative to MET/HY cells. These findings suggest that GJIC and Cx43 expression contribute to breast cancer cell adhesion and migration, possibly through a mechanism involving OB-cadherin, and these changes in turn regulate the metastatic potential of breast cancer cells, especially to bone.     

星形胶质细胞的缝隙连接蛋白43参与冷冻伤后脑水肿的形成

目的:探讨脑水肿后星形胶质细胞缝隙连接蛋白43(Cx43)的表达及其在脑水肿的发生发展过程中所起的作用。方法:采用颅骨外液氮冷冻法建立大鼠右侧顶叶皮层脑水肿模型。实验分为假手术组、脑水肿模型组和脑水肿模型+缝隙连接阻断剂(carbenoxolone或octanol)干预组。干湿重法测定冷冻伤后的脑含水量;甲酰胺法测定大鼠血脑屏障通透性的改变;HE染色观察冷冻伤脑组织的病理变化;Western blot法和免疫组化检测Cx43蛋白的表达情况。结果:冷冻伤可引起大鼠损伤脑皮层区的含水量增加,在冷冻伤后24 h脑水肿发展到高峰。冷冻伤引起损伤脑皮层区域的血脑屏障通透性增加,范围大于直接冷冻损伤区。HE染色观察显示冷冻伤中心区细胞坏死明显,而冷冻伤周围区域出现水肿。脑冷冻伤引起冷冻伤周围皮层区域的Cx43蛋白表达增加,但冷冻伤中心区的Cx43蛋白表达降低。Carbenoxolone或octanol阻断Cx43的功能,降低了冷冻伤皮层区的含水量和血屏障通透性。结论:脑水肿时星形胶质细胞上的Cx43表达上调,功能增强;阻断Cx43的功能可在一定程度上减轻脑水肿。     

Expression patterns of connexin43 protein during facial development in the chick embryo: Associations with outgrowth,attachment, and closure of the midfacial primordia

ABSTRACT Background: In a prior report, evidence was presented for the presence of gap junction proteins [connexin32 and connexin43 (Cx43)] in embryonic facial primordia. The purpose of the present study was, first, to examine in detail the patterns of distribution of Cx43 protein in embryonic chick facial primordia and, second, to consider the possible roles played by this protein during midfacial development. Methods: Chick embryo heads were serially sectioned and processed for immunofluorescent localization of Cx43. The developmental stages examined encompassed the period of formation, enlargement, and union of the facial primordia. Western blot analysis of the facial primordia was also performed. Results: Analysis of serial sections revealed the presence of signal in both epithelium and mesenchyme at sites of attachment in each of the midfacial primordia (i.e., the medial nasal, lateral nasal, and maxillary processes). Furthermore, although signal was concentrated in mesenchyme in the distal tips of the primordia at sites of attachment, immunoreactivity was absent, sparse, or less intense outside the areas of attachment. In some cases (i.e., the maxillary process), immunoreactive signal in mesenchyme did not appear in the distal tip until the primordia approximated each other or contact of the primordia was initiated. Most significantly, signal was also found between the facial primordia in nonprimordial epithelium and mesenchyme at sites where the primordia were joined. Conclusions: These data suggest that the expression of Cx43 protein is spatially and temporally regulated in the facial primordia and that the patterns of expression that were observed are significant to the cascade of events that ultimately lead to the attachment and union of the primordia that form the midface. Anat. Rec. 248:279-290, 1997. © 1997 Wiley-Liss, Inc.