Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.     

Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells

BACKGROUND: The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells. METHODS: Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed. RESULTS: Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation. CONCLUSIONS: Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.     

SDH基因在肾上腺嗜铬细胞瘤中的表达和意义

目的 研究抑癌基因线粒体琥珀酸脱氢酶(SDH)及其表达产物SDH蛋白在正常肾上腺髓质和肾上腺嗜铬细胞瘤中的表达,比较SDH mRNA的表达与SDH蛋白的表达关系.方法 取正常肾上腺髓质和嗜铬细胞瘤组织标本,应用逆转录--聚合酶链反应(RT-PCR)及免疫组织化学方法 检测SDH mRNA及SDH蛋白的表达情况.结果 RT-PCR显示,嗜铬细胞瘤组织和正常肾上腺髓质均可扩增出灰白色的SDH条带.免疫组化显示SDH蛋白在嗜铬细胞瘤中表达为75.8%,在正常肾上腺髓质表达为10%.结论 肾上腺嗜铬细胞瘤存在SDH基因突变,推测SDH基因与嗜铬细胞瘤的发生存在明显相关性.     

mRNA expression of topoisomerase II in human tumors and normal tissues.

The cellular levels of topoisomerase II expression were compared between 10 fresh human tumors and normal tissues to predict the selective anticancer effect of its inhibitors such as adriamycin and VP-16. Topoisomerase II expression was observed in 9 of the 10 tumor tissues (90.0 per cent), 3 of which showed extremely high levels, whereas only 5 of the normal tissues (50.0 per cent) expressed any cellular topoisomerase II and the levels were not higher than those seen in the cancer cells. Six of the 9 positive tumors showed a higher level of topoisomerase II expression than the normal tissues, while the other 3 showed the same level. It can be interpreted from these results that topoisomerase II inhibitors could be effective in cancer patients due to the greater level of this enzyme in tumor cells than in normal tissues. Thus, it is suggested that a comparative analysis of topoisomerase II expression between tumors and normal tissues may be useful for predicting the selective cytotoxicity of topoisomerase II inhibitors in clinical practice.     

肝癌缺失基因在胃癌中的表达及启动子甲基化

目的探讨肝癌缺失基因(DLC1)与胃癌及其临床分期、分化程度等关系,DLC1mRNA表达与启动子甲基化的关系。方法用半定量逆转录聚合酶链反应方法检测34例原发性胃癌及癌旁正常组织中DLC1mRNA表达;用甲基化特异聚合酶链反应法(MSP)检测启动子甲基化。结果正常组织中DLC1mRNA均正常表达,胃癌组织DLC1mRNA低表达或表达缺失;正常组织0.62±0.11,胃癌组织0.24±0.17,差异有统计学意义(P<0.01)。DLC1mRNA表达与淋巴结转移和肿瘤分化程度有关,与性别、肿瘤大小和临床分期无关。正常组织中未发现DLC1基因启动子甲基化;胃癌组织中DLC1启动子甲基化阳性12例,甲基化率35.3%。启动子甲基化与无甲基化患者间DLC1mRNA表达差异有统计学意义(P<0.01)。结论DLC1与胃癌存在明显相关性,是重要的抑癌基因。启动子甲基化与DLC1mRNA表达抑制密切相关,可能是影响DLC1在胃癌中低表达的最主要因素,DLC1是胃癌甲基化谱中主要成员。     

新肿瘤抑制基因SLC5A8在结直肠癌组织中mRNA转录水平的研究

目的 分析新肿瘤抑制基因SLC5A8在结直肠癌组织中mRNA的转录水平.方法 收集23例结直肠癌患者标本,分别切取癌组织及癌旁组织,采用实时荧光定量PCR（RT-qPCR）技术检测基因SLC5 A8的mRNA转录水平,并对癌组织和癌旁组织的结果进行t检验分析.结果 结直肠癌组织中SLC5 A8基因的mRNA转录水平显著低于癌旁组织（P=0.002）.结论 SLC5A8基因在结直肠癌组织中表达下降或缺失,提示其与结直肠癌的发生存在一定关系.     

mRNA expression of topoisomerase II in human tumors and normal tissues

The cellular levels of topoisomerase II expression were compared between 10 fresh human tumors and normal tissues to predict the selective anticancer effect of its inhibitors such as adriamycin and VP-16. Topoisomerase II expression was observed in 9 of the 10 tumor tissues (90.0 per cent), 3 of which showed extremely high levels, whereas only 5 of the normal tissues (50.0 per cent) expressed any cellular topoisomerase II and the levels were not higher than those seen in the cancer cells. Six of the 9 positive tumors showed a higher level of topoisomerase II expression than the normal tissues, while the other 3 showed the same level. It can be interpreted from these results that topoisomerase II inhibitors could be effective in cancer patients due to the greater level of this enzyme in tumor cells than in normal tissues. Thus, it is suggested that a comparative analysis of topoisomerase II expression between tumors and normal tissues may be useful for predicting the selective cytotoxicity of topoisomerase II inhibitors in clinical practice.     

The distribution of calcium in normal and tetracycline-modified bones of developing chick embryos

The presence of calcium (GBHA technique) in the periosteal cells, osteoblasts, osteocytes and chondrocytes of developing chick embryo bone confirms the findings ofKashiwa (1966) in the rat. Modification of bone development by tetracycline administration indicates that there is a direct correlation between the amount of calcium within these cells and the degree of mineralization of the adjacent matrices. The calcium of the calcification front of bone and the abnormally calcified cartilage matrix resulting from tetracycline treatment appears to differ from that of the older bone and normally calcifying cartilage matrix in its ability to chelate with GBHA. The distribution of the GBHA positive material appears to correspond to that of a pyridine-resistant bound lipid previously reported.

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Zusammenfassung Das Vorkommen von Calcium (GBHA-Technik) in den Zellen des Periosts, in den Osteoblasten, Osteocyten und Chondrocyten des sich entwickelnden Knochens im Hühnerembryo bestätigt die Resultate, welcheKashiwa (1966) bei der Ratte beschrieb. Eine durch Tetracyclingaben hervorgerufene Veränderung der Knochenentwicklung zeigt, daß eine direkte Korrelation zwischen dem Calciumgehalt in diesen Zellen und dem Mineralisationsgrad der sie umgebenden Matrices besteht. Das Calcium an der Calcificationsgrenze des Knochens und in der durch Tetracyclinbehandlung abnormal verkalkten Knochenmatrix scheint sich dadurch zu unterscheiden von älteren Knochen und von normal verkalkender Knorpelmatrix, daß es mit GBHA ein chelat zu bilden vermag. Die Verteilung von GBHA-positivem Material scheint mit der eines früher beschriebenen pyridinresistenten gebundenen Lipids übereinzustimmen.

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Résumé La présence de calcium (technique GBHA) dans les cellules du périoste: ostéoblastes, ostéocytes et chondrocytes d'os en cours de développement chez l'embryon de poulet, confirme les résultats deKashiwa (1966) chez le rat. La modification du développement osseux par l'administration de tétracycline indique qu'il y a une corrélation directe entre la quantité de calcium à l'intérieur de ces cellules et le degré de minéralisation des matrices adjacentes. Le calcium du front de calcification de l'os et de la matrice du cartilage anormalement calcifiée qui résulte du traitement à la tétracycline ne semble pas avoir la même capacité à entrer en chélation sous l'action du GBHA, que le calcium d'os plus âgé et de matrice normalement calcifiée. La distribution du matériel positif au GBHA semble correspondre à celle d'un lipide lié, résistant à la pyridine, et déjà décrit.

     

Aquaporin-4 expression in adult and developing mouse and rat kidney

Aquaporin-4 (AQP4) is a member of the aquaporin water-channel family. AQP4 is expressed primarily in the brain, but it is also present in the collecting duct of the kidney, where it is located in the basolateral plasma membrane of principal cells and inner medullary collecting duct (IMCD) cells. Recent studies in the mouse also have reported the presence of AQP4 in the basolateral membrane of the proximal tubule. The purpose of this study was to establish the pattern of AQP4 expression during kidney development and in the adult kidney of both the mouse and the rat. Kidneys of adult and 3-, 7-, and 15-d-old mice and rats were preserved for immunohistochemistry and processed using a peroxidase pre-embedding technique. In both the mouse and the rat, strong basolateral immunostaining was observed in IMCD cells and principal cells in the medullary collecting duct at all ages examined. Labeling was weaker in the cortical collecting duct and the connecting tubule, and there was no labeling of connecting tubule cells in the mouse. In adult mouse kidney, strong AQP4 immunoreactivity was observed in the S3 segment of the proximal tubule. However, there was little or no labeling in the cortex or around the corticomedullary junction in 3- and 7-d-old mice. Between 7 and 15 d of age, distinct AQP4 immunoreactivity appeared in the S3 segment of the mouse proximal tubule concomitant with the differentiation of this segment of the nephron. Labeling of proximal tubules was never observed in the rat kidney. These results suggest that there are differences in transepithelial water transport between mouse and rat or that additional, not yet identified water channels exist in the rat proximal tubule.     

Yes-相关蛋白和大肿瘤抑制基因1在肾透明细胞癌中的表达及意义

目的 探讨Yes-相关蛋白(Yes-associated protein,YAP)和大肿瘤抑制基因1(large tumor suppressor gene 1,LATS1)在肾透明细胞癌(renal clear cell carcinoma,RCCC)组织中的表达及意义. 方法 RCCC标本30例,男15例,女15例.年龄36～77岁,中位年龄63岁.Ⅰ～Ⅱ期17例,Ⅲ～Ⅳ期13例.高分化8例,中分化13例,低分化9例.采用逆转录聚合酶链反应及免疫组化技术检测30例RCCC组织及相应正常肾组织中YAP、LATS1 mRNA及其蛋白的表达,结合临床资料进行分析. 结果 RCCC组织中YAP mRNA表达量为0.569±0.066,正常肾组织为0.515±0.068,组间差异有统计学意义(P =0.003);LATS1 mRNA表达量分别为0.454±0.115、0.514±0.093,组间差异有统计学意义(P =0.029).YAP蛋白表达阳性率分别为63.3％和33.3％,组间差异有统计学意义(P =0.020);LATS1蛋白表达阳性率分别为46.7％和76.7％,组间差异有统计学意义(P=0.017).高、中、低分化组织中YAP蛋白表达阳性率分别为33.3％、61.5％、88.9％,组间差异有统计学意义(P=0.018);LATS1蛋白表达阳性率分别为75.0％、53.8％、11.1％,组间差异有统计学意义(P=0.024).Ⅰ～Ⅱ、Ⅲ～Ⅳ期组织中YAP蛋白表达阳性率分别为47.1％、84.6％,组间差异有统计学意义(P =0.034);LATS1蛋白表达阳性率分别为64.7％、23.1％,组间差异有统计学意义(P =0.024). 结论 YAP与LATS1在RCCC发生和发展过程中起重要作用,有望成为RCCC治疗的新靶点之一.     

Denervation and reinnervation of adult skeletal muscle modulate mRNA expression of neuregulin‐1 and ErbB receptors

Skeletal muscle atrophy represents one of the main causes of poor outcome of microsurgical nerve reconstruction. Recent studies have pointed to the importance of the neuregulin/ErbB signaling pathway in the development and regeneration of the neuromuscular system. Here, we show by immunohistochemistry, RT‐PCR, and Western blotting analyses, in an in vivo model of adult skeletal muscle denervation/reinnervation, that expression of Neuregulin1 (NRG1) and ErbB receptors is regulated by the innervation condition. We found out that a significant upregulation of the α‐, but not β‐, isoform of NRG1, as well as of ErbB2, ErbB3, and ErbB4‐cyt1 isoform occurs as a consequence of denervation of flexor digitorum muscles of the rat forelimb by median nerve transection. Moreover, after tubulization median nerve repair, and consequent muscle reinnervation, all messengers of the NRG1/ErbB system are promptly downregulated. Therefore, our results suggest the existence of a α‐NRG1‐mediated autocrine and/or paracrine trophic loop in skeletal muscles that is activated after denervation and promptly deactivated after nerve reconstruction. This myotrophic loop is a promising therapeutic target for the prevention of muscle atrophy. Yet, the recent demonstration of a similar α‐NRG1‐mediated gliotrophic loop in denervated Schwann cells provides a possible explanation for the effectiveness of muscle conduits for tubulization nerve repair. © 2009 Wiley‐Liss, Inc. Microsurgery, 2009.     

Von Hippel-Lindau tumor suppressor protein and hypoxia-inducible factor in kidney cancer

The development of hereditary von Hippel-Lindau (VHL) disease and the majority of sporadic kidney cancers are due to the functional inactivation of the VHL gene. The product of the VHL gene, pVHL, in association with elongins B and C, cullin 2, and Rbx1 form an E3 ubiquitin-ligase complex VEC that targets the alpha subunits of hypoxia-inducible factor (HIF) for ubiquitination. Ubiquitin-tagged HIF-alpha proteins are subsequently degraded by the common 26S proteasome. pVHL functions as the substrate-docking interface that specifically recognizes prolyl-hydroxylated HIF-alpha. This hydroxylation occurs only in the presence of oxygen or normoxia. Thus, under hypoxia, HIF-alpha subunits are no longer subjected to degradation and are thereby able to dimerize with the common and constitutively stable beta subunits. The heterodimeric HIFs upregulate a myriad of hypoxia-inducible genes, triggering our physiologic response to hypoxia. Inappropriate accumulations of HIF-alpha in VHL disease are believed to contribute to the pathogenesis via the upregulation of several of these HIF target genes. Our current molecular understanding of the roles of HIF and pVHL in the development of VHL-associated clear-cell renal cell carcinoma (CC-RCC) is the focus of this review.     

Effect of ascorbic acid on protein synthesis and collagen hydroxylation in continuous flow organ cultures of adult mouse periodontal tissues

Summary A continuous flow organ culture system (CFCS) was used to determine the effect of ascorbic acid on the synthesis of collagen and noncollagenous protein by bone of the alveolar process and periodontal ligament in organ cultures of adult mouse periodontium. For the last 24 h of 2 day cultures, 5 μCi/ml³H-proline was added to the medium. Highly purified collagenase was used to separate the collagenous and noncollagenous proteins and the incorporation of isotope into each fraction measured. Collagen synthesized in the presence of less than 10 μg/ml ascorbic acid was found to be highly under-hydroxylated (pro:hypro sp. acts. 2.3–3.1) in both tissues. When the ascorbic acid levels were between 25 and 100 μg/ml, the synthesis of collagenous proteins was selectively stimulated and hydroxylation significantly improved (pro:hypro sp. acts 1.72–1.89). The effect of ascorbic acid was not related to tissue viability since tissues cultured initially in the absence of ascorbic acid were able to recover completely when compared to controls given ascorbic acid continuously. The proportion of radioactivity in collagen and noncollagenous protein, collagen hydroxylation, and percentage of collagen synthesized as type III (av. 23%) in bone of the alveolar process was similar to that found in vivo. However, in the periodontal ligament in vitro the proportion of noncollagenous protein synthesized was increased from 70% to 87% and the percentage of type III collagen increased from 14% to 26% compared to in vivo results.