Antagonistic Effects of Insulin and TGF-ß3 during Chondrogenic Differentiation of Human BMSCs under a Minimal Amount of Factors.

Growth factors are crucial regulators of cell differentiation towards tissue and organ development. Insulin and transforming growth factor-ß (TGF-ß) have been used as the major factors for chondrogenesis in vitro, by activating the AKT and Smad signaling pathways. Previous reports demonstrated that AKT and Smad3 have a direct interaction that results in the inhibition of TGF-ß-mediated cellular responses. However, the result of this interaction between AKT and Smad3 during the chondrogenesis of human bone marrow-derived stem/progenitor cells (hBMSCs) is unknown. In this study, we performed functional analyses by inducing hBMSCs into chondrogenesis with insulin, TGF-ß3 or in combination, and found that TGF-ß3, when applied concomitantly with insulin, significantly decreases an insulin-induced increase in mRNA levels of the master regulator of chondrogenesis, SOX9, as well as the regulators of the 2 major chondrocyte markers, ACAN and COL2A1. Similarly, the insulin/TGF-ß3-treated group presented a significant decrease in the deposition of cartilage matrix as detected by safranin O staining of histological sections of hBMSC micromass cultures when compared to the group stimulated with insulin alone. Intracellular analysis revealed that insulin-induced activation of AKT suppressed Smad3 activation in a dose-dependent manner. Accordingly, insulin/TGF-ß3 significantly decreased the TGF-ß3-induced increase in mRNA levels of the direct downstream factor of TGF-ß/Smad3, CCN2/CGTF, compared to the group stimulated with TGF-ß3 alone. On the other hand, insulin/TGF-ß3 stimulation did not suppress insulin-induced expression of the downstream targets TSC2 and DDIT4/REDD1. In summary, insulin and TGF-ß3 have antagonistic effects when applied concomitantly, with a minimal number of factors. The application of an insulin/TGF-ß3 combination without further supplementation should be used with caution in the chondrogenic differentiation of hBMSCs.

Age-dependent changes in the susceptibility to thiopental anesthesia in mice: analysis of the relationship to the functional expression of GABA transporter.

The potency of anesthetics changes during development, probably due not only to pharmacokinetic factors such as differential distribution and/or metabolism, but also to pharmacodynamic factors such as changes to the GABAergic system in the brain. To explore the latter mechanism, we focused on the GABA transporter (GAT), the uptake system for GABA, which participates in the synaptic clearance of GABA. Thiopental-induced anesthesia, as assessed by the onset and duration of loss of the righting reflex, was more pronounced in 3-week-old mice than in 7-week-old mice. Both NO-711 and SKF89976A, selective GAT-1 inhibitors, significantly enhanced the anesthesia in the 7-week-old but not in the 3-week-old mice. In synaptosomes prepared from the cerebral cortex, the kinetics of GABA transport was similar between the two age groups, as assessed by [(3)H]GABA uptake assay. In addition, expression of GAT mRNA was similar between the two age groups, as assessed by quantitative RT-PCR. Thiopental reduced [(3)H]GABA uptake only at high concentrations in a similar manner at both ages. Conversely, the ability of SKF89976A to inhibit [(3)H]GABA uptake was greater in the 7-week-old mice than in the 3-week-old mice. Based on these results, GAT seems unlikely to contribute to the greater susceptibility to thiopental anesthesia in 3-week-old mice, while the increased ability of GABA uptake inhibitors to enhance thiopental-induced anesthesia in 7-week-old mice is at least partly due to higher sensitivity of GAT to the inhibitors.