In PC Cl3 cells, a rat thyroid cell line, angiotensin (Ang II) activates the atypical protein kinase C-ζ (PKC-ζ) and the extracellular signal-regulated kinase (ERK) pathways. We here studied the Ang II effects on PC Cl3 cell proliferation. It was found that Ang II: (1) induced the phosphorylation of protein kinase B (PKB), (2) induced the growth-related early gene c-fos expression, (3) enhanced the cyclin E and p27kip expression, (4) had no effects on Cdk2, and (5) did not affect the transition from G0/G1 to S phase. Inhibition of phosphoinositide-3kinase by LY294002 further increased the effect of Ang II on p27kip induction, whilst PKCs inhibition by GF109203X decreased such effect. The role of PKC-ζ was recognized by the use of a synthetic myristoylated peptide with sequences based on the endogenous PKC-ζ pseudosubstrate and by PKC-ζ downregulation using the small interfering RNA (siRNA). Insulin had a replicating effect on PC Cl3 cells, induced the phosphorylation of PKB, decreased p27kip expression and had no effect on the PKC-ζ cytosol-to-membrane translocation. PC Cl3 cell proliferation was induced more potently by simultaneous stimulation with insulin and Ang II than by stimulation with insulin alone, and the effect on p27kip expression was similar to that obtained with insulin only. These observations demonstrate that in PC Cl3 cells Ang II causes a block in G1 phase, although both ERK and PKB pathways are activated, and this effect may be due to the upregulation of p27kip and PKC-ζ operativity.
Simona Romano, Antonella Muscella, Carlo Storelli and Santo Marsigliante
Luca Ulianich, Maria Giovanna Elia, Antonella Sonia Treglia, Antonella Muscella, Bruno Di Jeso, Carlo Storelli and Santo Marsigliante
In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.