ABSTRACT
Protein kinase C (PKC) activity was measured in rat FRTL-5 cells cultured in medium containing insulin and transferrin (2H medium), in cells treated with phorbol 12,13-dibutyrate (PDBu), an activator of PKC, and in cells treated with 4α-12,13-phorbol didecanoate (4α-PDD), an inactive phorbol ester, in order to study the translocation and down-regulation of its activity. Provided the PDBu induced translocation and down-regulation of PKC activity, we used the down-regulated cells to study the roles played by PKC in DNA synthesis induced by stimuli which increase cyclic AMP (cAMP) and a cAMP analogue. Cytosolic preparations obtained from cells cultured in 2H media were fractionated on a DEAE-cellulose column, and fractions were assayed for PKC activity. PKC activity was eluted at concentrations between 0·06 and 0·15 mol NaCl/l with a peak at 0·1 mol/l. Exposure of cells to PDBu (100 ng/ml) resulted in 53% loss of initial PKC activity in the cytosolic fraction in concert with reciprocal augmentation of PKC activity in the particulate fraction that peaked at 321% of the initial level at 15 min of exposure.
When cells were stimulated with 100 ng PDBu/ml for 15 min, three- and 1·9-fold greater PKC activity appeared in the particular and total cellular (cytosol plus particulate) fractions respectively than was the case for stimulation with 10 ng PDBu/ml. The total cellular PKC activity transiently increased at 15 min to 137% and subsequently decreased to 74, 40 and 25% of the initial level at 1, 12 and 24 h respectively. However, translocation of PKC activity to the particulate fraction was not observed in cells treated for 15 min with either 10 or 100 ng 4α-PDD/ml and total cellular PKC activity was modestly reduced, to 97% of the initial level in cells exposed to 4α-PDD (100 ng/ml) for 24 h.
[3H]Thymidine incorporation was measured in cells whose PKC activity was stimulated by TSH, forskolin or 8-bromoadenosine cAMP simultaneously with PDBu (100 ng/ml), and also in cells whose PKC activity had been down-regulated by pretreatment with PDBu prior to the addition of each stimulant. The [3H]thymidine incorporation induced by each of the stimulants was inhibited in cells whose PKC activity was subjected to simultaneous stimulation, but was enhanced in cells whose PKC activity had previously been depleted. This potentiating effect of PDBu pretreatment on subsequent TSH-induced [3H]thymidine incorporation was not observed in cells exposed to PDBu for less than 12 h, but was observed in cells exposed for 24 h or longer.
These findings support the hypothesis that PKC regulates TSH-induced DNA synthesis in an inhibitory fashion at sites both proximal and distal to the cAMP generation process in FRTL-5 cells, and that a critical amount of activity must be present for this inhibition to occur.
Journal of Endocrinology (1993) 138, 379–389
3P189 Movement of nucleus in cells cultured on micro-patterned substrates(Cell biology,Poster,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))