This study investigates the mechanism through which increased 30K protein inhibits ecdysone‐induced apoptosis in the B m5 silkworm ovarian cell line. Treatment of Bm5 cells with 20‐hydroxyecdysone (20 E ) after transfection with the p IZT / V 5‐ H is control vector triggered apoptosis, but 20 E treatment did not trigger apoptosis in B m5 cells transfected with the p IZT /30 K / V 5‐ H is vector. To confirm its inhibitory effect on apoptosis, 30 K protein was first purified from Escherichia coli transformed with a 30 K expression vector and used to generate specific antibodies in mice. Anti‐30 K antiserum was used to confirm synthesis of the 30 K protein in p IZT /30 K / V 5‐ H is‐transfected B m5 cells and to detect 30 K protein binding to the ecdysone receptor‐ B 1 ( E c R ‐ B 1). Anti‐30 K antiserum was used to immunoprecipitate protein complexes containing 30 K from B m5 cells transfected with p IZT /30 K / V 5‐ H is vector and treated with 20 E . We observed that 30 K proteins bound primarily to the E c R ‐ B 1 and not to ultraspiracle ( USP ). Reciprocal immunoprecipitation of E c R ‐ B 1‐containing complexes from B m5 cells transfected with control p IZT / V 5‐ H is vector and treated with 20 E showed that E c R ‐ B 1 bound to USP in the absence of 30 K but did not bind to USP in p IZT /30 K / V 5‐ H is‐transfected B m5 cells. These results demonstrate that 30 K proteins block USP binding to E c R ‐ B 1 through formation of a 30 K / E c R ‐ B 1 complex, resulting in inhibition of 20 E ‐induced B m5 cell apoptosis.
The Wnt family of proteins regulates development and cell growth. We identified Wnt3a-based regulatory mechanisms for cell proliferation in NIH3T3 fibroblast cells. The degree of Wnt3a-induced proliferation was reduced by beta-catenin small interfering RNA (siRNA) and extracellular signal-regulated kinase (ERK) siRNA, indicating that both the ERK and Wnt/beta-catenin pathways are involved in Wnt3a-induced proliferation. Wnt3a immediately and transiently activated the Raf-1-MEK-ERK cascade in a manner distinct from that of the beta-catenin increase seen in cells treated with Wnt3a. Wnt3a-induced ERK activation was maintained even though basal ERK activities were reduced by beta-catenin siRNA, indicating that Wnt3a may activate the ERK pathway independently of beta-catenin. The ERK pathway was however, activated by beta-catenin transfection, which was abolished by co-transfection with dominant-negative Tcf-4. Therefore, ERK pathway activation by Wnt signaling could occur at multiple levels, including beta-catenin-independent direct signaling resulting from a Wnt3a and beta-catenin/Tcf-4-dependent post gene transcriptional event. Wnt3a stimulated the G1 to S phase cell cycle progression. This stimulation was reduced by the ERK pathway inhibitor, indicating that Wnt3a promotes proliferation by stimulating the ERK pathway. Wnt3a therefore stimulates the proliferation of fibroblast cells, at least in part, via activation of the ERK and Wnt/beta-catenin pathways.
We have developed and evaluated a novel perfusion flow bioreactor system which facilitates the observation of living cells for extended periods of time. Cell viability showed that cell growth rate cultured (without medium perfusion) using the novel flow perfusion chamber system revealed a similar trend (p>0.05) compared to that of static culture. Also, the cell viability under the static culture (without perfusion) and flow perfusion culture (flow rate: 0.03 ml/min) with the developed perfusion bioreactor system was compared. The cell growth rate of the flow perfusion culture was significantly higher (p<0.05) than that of static culture for 4 days. The cell growth rate was efficiently increased to about 10-15% with the precision perfusion culture. These results indicate that a bioreactor system that can provide mechanical signals has important applications. Additionally, the developed cell culture chamber system has been successfully used to maintain and record the morphology of cultured alveolar bone marrow stem cells. It was possible to monitor and capture the live cell morphology cultured in the perfusion bioreactor. The characteristics of the bioreactor developed in this study was fast to culture stem cells with mechanical stimulation and to monitor live cell imaging while promoting healthy cellular activity outside of an incubator environment. Through experimental results, the developed perfusion bioreactor could increase cell growth with proper flow based on mechanical stimuli.
In this study, the properties and biocompatibilities of composite scaffolds were investigated to develop an appropriate scaffold for dental tissue regeneration using human dental pulp stem cells. Composite scaffolds consisted of poly-D,L-lactic acid (P(D,L)LA) and bioceramic materials of tooth powders fabricated using sintered teeth. Highly porous composite bioceramic scaffolds promoted biocompatibility and had the interconnected pores that could adequately support cell adhesion and proliferation. Also, we investigated the properties and biocompatibilities of composite scaffolds prepared by a solvent casting/particulate leaching method and a gas forming method. We prepared the composite ceramic scaffold using toothapatites obtained from extracted teeth, and assessed the biocompatibility of the composite scaffolds by the two methods in order to find out the most appropriate scaffolds for tooth regeneration. The human dental pulp stem cells seeded on the composite scaffolds were easily attached and well proliferated as confirmed by a cytotoxicity test, cell adhesion assessment and histological study. Through experimental results, composite bone scaffolds were able to be proven as appropriate scaffolds for dental tissue regeneration using human dental pulp stem cells.
The platelet-rich fibrin (PRF) is known as a rich source of autologous cytokines and growth factors and universally used for tissue regeneration in current clinical medicine. However, the microstructure of PRF has not been fully investigated nor have been studied the key molecules that differ PRF from platelet-rich plasma. We fabricated PRF under Choukroun's protocol and produced its extract (PRFe) by freezing at -80°C. The conventional histological, immunohistological staining, and scanning electron microscopy images showed the microstructure of PRF, appearing as two zones, the zone of platelets and the zone of fibrin, which resembled a mesh containing blood cells. The PRFe increased proliferation, migration, and promoted differentiation of the human alveolar bone marrow stem cells (hABMSCs) at 0.5% concentration in vitro. From the results of proteome array, matrix metalloproteinase 9 (MMP9) and Serpin E1 were detected especially in PRFe but not in concentrated platelet-rich plasma. Simultaneous elevation of MMP9, CD44, and transforming growth factor β-1 receptor was shown at 0.5% PRFe treatment to the hABMSC in immunoblot. Mineralization assay showed that MMP9 directly regulated mineralization differentiation of hABMSC. Transplantation of the fresh PRF into the mouse calvarias enhanced regeneration of the critical-sized defect. Our results strongly support the new characteristics of PRF as a bioscaffold and reservoir of growth factors for tissue regeneration.
Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.Alternative splicing gives a variation from the same gene.ResultsA novel alternative splicing form of Met exists in human skeletal muscle, and its depletion decreases muscle differentiation, whereas its overexpression acts oppositely.ConclusionA novel alternative splicing form of Met regulates skeletal muscle differentiation.SignificanceThis is an example of regulation by alternative splicing, which affects skeletal muscle differentiation. Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.Alternative splicing gives a variation from the same gene. A novel alternative splicing form of Met exists in human skeletal muscle, and its depletion decreases muscle differentiation, whereas its overexpression acts oppositely. A novel alternative splicing form of Met regulates skeletal muscle differentiation.
We show here that expression of chromogranins in non-neuroendocrine NIH3T3 cells significantly increased the amount of IP3-mediated intracellular Ca2+ mobilization in these cells, whereas suppression of them in neuroendocrine PC12 cells decreased the amount of mobilized Ca2+. We have therefore investigated the relationship between the IP3-induced intracellular Ca2+ mobilization and secretory granules. The level of IP3-mediated Ca2+ release in CGA-expressing NIH3T3 cells was 40% higher than in the control cells, while that of CGB-expressing cells was 134% higher, reflecting the number of secretory granules formed. Suppression of CGA and CGB expression in PC12 cells resulted in 41 and 78% reductions in the number of secretory granules, respectively, while the extents of IP3-induced Ca2+ release in these cells were reduced 40 and 69%, respectively. The newly formed secretory granules of NIH3T3 cells contained all three isoforms of the IP3Rs. Comparison of the concentrations of the IP3R isoforms expressed in the ER and nucleus of chromogranin-expressing and nonexpressing NIH3T3 cells did not show significant differences, indicating that chromogranin expression did not affect the expression of endogenous IP3Rs. Nonetheless, the IP3R concentrations in secretory granules of chromogranin-expressing NIH3T3 cells were 3.5−4.7-fold higher than those of the ER, similar to the levels found in secretory granules of neuroendocrine chromaffin cells, thus suggesting that the IP3Rs targeted to the newly formed secretory granules are newly induced by chromogranins without affecting the expression of intrinsic IP3Rs. These results strongly suggest that the extent of IP3-induced intracellular Ca2+ mobilization in secretory cells is closely related to the number of secretory granules.
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