三维回转骨细胞条件培养基对成骨细胞功能的调节作用

骨细胞是骨组织中主要的力学感受器.研究失重条件下骨细胞对效应细胞的调控作用对于揭示失重引起的骨丢失机制具有重要意义.本研究拟采用三维回转器模拟失重,探讨模拟失重骨细胞条件培养基(RCM)对成骨功能的调节作用.小鼠骨细胞系MLO-Y4三维回转培养72h后,收集回转条件培养基(RCM)和未回转对照组的条件培养基(CCM),用四甲基偶氮唑盐比色(MTT)法、对硝基苯磷酸(pNPP)法和流式细胞术(FCM)分别检测RCM对小鼠成骨样细胞系MC3T3-E1增殖、周期及细胞分泌碱性磷酸酶(ALP)活性的影响.采用RT-PCR方法检测RCM对MC3T3-E1成骨相关基因表达的影响.结果显示,三维随机回转72h后的MLO-Y4RCM可促进MC3T3-E1增殖:条件培养基培养MC3T3-E124h和48h后,50%RCM组比CCM组分别增加了1.62和1.60倍,差异显著(*P0.05),培养72h后,100%RCM组比CCM组增加了1.69倍,差异显著(*P0.05);细胞周期检测结果表明,条件培养基培养24、48和72h后,RCM组部分恢复CCM引起的MC3T3-E1细胞周期阻滞;MC3T3-E1的ALP活性在RCM组和CCM组之间无差异;RT-PCR检测结果表明,100%MLO-Y4条件培养基培养MC3T3-E148h后,降低了成骨相关基因ALP、Runx2、OPN、OC的表达.差异显著(*P0.05,**P0.01,***P0.001).实验结果表明,三维随机回转模拟失重培养骨细胞72h后的条件培养基促进了成骨细胞增殖,抑制了成骨相关基因表达.     

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.     

Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most subapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression.     

Biochemical and immunological approaches to the study of gap junctional communication

Summary Studies on gap junctions isolated from rat liver by a procedure that avoids exogenous proteolysis (Hertzberg, E. L.; Gilula, N. B.; J. Biol. Chem. 254: 2138–2147; 1979) are described. The original isolation procedure was modified to increase the yield and has been extended to the preparation of gap junctions from mouse and bovine liver. Peptide map studies showed that the 27,000-dalton polypeptides present in liver gap junction preparations from all three sources are homologous and are not derived from other polypeptides of higher molecular weight that are observed in cruder preparations. Similar studies with lens fiber junctions demonstrated no homology between liver and lens junction polypeptides. Antibodies to the lens junction polypeptide did not cross-react with the liver gap junction polypeptide, further supporting this conclusion. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center. Research in the laboratory was supported by grants to Dr. Gilula from the National Institute of Health (HL 16507 and GM 24753).     

Mechanical Loading Stimulates Expression of Connexin 43 in Alveolar Bone Cells in the Tooth Movement Model

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.     

Comparison of Connexin 43, 40 and 45 Expression Patterns in the Developing Human and Mouse Hearts

The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.     

Control of Intracellular Movement of Connexins by E-Cadherin in Murine Skin Papilloma Cells

The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 cells and appear at cell-cell contact areas only in high-calcium medium. While Cx43 is unphosphorylated in P3E1 cells in low-calcium medium, two phosphorylated bands appeared at high calcium. However, when Cx26, which has no C-terminal tail that can undergo phosphorylation, was expressed in P3E1 cells, this connexin also moved to the plasma membrane after the calcium shift and partly colocalized with Cx43, suggesting that C-terminal phosphorylation is not essential for E-cadherin-mediated intracellular transport of connexins. In low calcium, both Cx26 and Cx43 remained and colocalized in the endoplasmic reticulum. As early as 30 min after the shift to high-calcium medium, both Cx43 and Cx26 began to accumulate in the Golgi apparatus. Intracellular movement of connexins to the cytoplasmic membrane at high calcium was effectively blocked by cytochalasin D and brefeldin A. These results suggest that E-cadherin junction formation at high calcium leads to formation of actin cables, which directly or indirectly transport connexins from the cytoplasm to the cell-cell contact membranes via the Golgi apparatus.     

Measurement of gap junctional communication by fluorescence activated cell sorting

Summary Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.     

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein,in mammal heart and brain

Summary According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314–322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrathin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions.The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43.Proteins ofM _r 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.     

Connexins 26, 32 and 43 are expressed in virgin, pregnant and lactating mammary glands

Gap junctions (GJ) are formed by a number of homologous proteins termed connexins. Here expression of connexins Cx26, Cx32 and Cx43, was evaluated by immunofluorescence (IF) in mammary glands from virgin, pregnant and lactating rats. Cx26, Cx32 and Cx43 labeling was detected in epithelial parenchymal cells at all functional stages. Cx26 and Cx32 labeling was very low in glands from virgin animals, somewhat greater in glands from pregnant animals and significantly higher (in number and size) in lactating animals. In the last ones, Cx26 and Cx32 punctate labeling was localized to the basal and lateral membranes of alveolar epithelial cells and collecting ductules. Cx43 punctate labeling was restricted to the periphery of alveoli towards the basal pole of epithelial cells at all functional stages, and it enlarged slightly during lactation. At this localization, Cx43 may form GJ between myoepithelial cells and/or between epithelial and myoepithelial cells. Cx43 was also found to be steadily expressed in the connective tissue which surrounds and invades each parenchymal lobe, at all functional stages. At this localization, Cx43 may couple fibroblasts and/or adipose cells. IF studies in sections from lactating mice showed the same distribution of connexins. Immunoblots confirmed specificity of labeling and the presence of Cx32 and Cx43 in the mammary gland. The increase in connexin expression detected during pregnancy and lactation may be important for epithelial cell differentiation and secretion in the mammary gland.     

Cross-linking of cardiac gap junction connexons by thiol/disulfide exchanges

Summary SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100mm) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.The abbreviations used are BSA bovine serum albumin - EDTA ethylene diamine tetra-acetic acid - IAA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsfonyl fluoride - SDS sodium dodecyl sulfate - Tris trishydroxymethyl-aminomethane