[Changes in the contractility of the NIH3T3 fibroblast induced by the over-expression of CPI-17]

The role of protein kinase C (PKC) in the regulation of contraction has been controversial. Recently, CPI-17, a PKC-potentiated inhibitor protein of PP1, has been cloned and shown to be specifically expressed in SMC. In this study, we over-expressed CPI-17 and its mutants in NIH3T3 cells, which do not express CPI-17, and examined its effect on the contractile property. For the measurement of tension, NIH3T3 cells were collected by trypsinization, mixed with type I collagen and made into a ring preparation (reconstituted ring: RR). The isometric tension developments were measured using this RR and a force transducer. The application of phorbol dibutyrate (PDBu; 0.3 microM) to RR transfected by CPI-17 induced a contraction that reached 2-3 times greater that the 10% FBS-induced contraction, while PDBu relaxed the RR transfected with vector alone (control) or CPI-17 mutants (T38A and T38E). The PDBu-induced contraction of the CPI-17 transfected RR could be inhibited by 3 microM GF109203X (PKC inhibitor) but not by 3 microM Y27632 (rho kinase inhibitor). The application of PDBu during contraction induced by 1 microM bradykinin induced further contraction in CPI-17 transfected RR, while it induced a complete relaxation in control RR. These results indicated that CPI-17 is a molecular switch that reverses the PKC mediated effect on contraction. The limited expression of CPI-17 to SMC may explain the previous observation that the effects of PKC stimulation were quite different in SMC from those of other cell types.