Protein expression of the epsilon subspecies of protein kinase C ceases as Swiss 3T6 fibroblasts increase in cell density even though message for the protein is still present

AbstractWe have noted previously that growth of C6 glioma cells from low cell density to confluency and quiescence in serum is accompanied by changes in protein content of different protein kinase C (PKC) subspecies. Here we show that the same occurs as non-contact-inhibiting Swiss 3T6 fibroblasts grow to high density in the presence of serum. Protein expression of PKC subspecies α and δ increases as the cells increase in density while that of PKC-ζ remains the same. Unusally, protein expression of PKC-ϵ is completely down-regulated as cells grow beyond about 50% confluency and no PKC-ϵ protein can be detected in 3T6 fibroblasts at high density by Western blotting. However, mRNA for PKC-ϵ is expressed at all stages of fibroblast growth as revealed by RT-PCR. When high-density 3T6 fibroblasts are passaged to low density in fresh medium, re-expression of PKC-ϵ protein is observed within 15 min and becomes down-regulated again as cells become more dense. This very rapid synthesis of PKC-ϵ is not blocked by the transcription inhibitor actinomycin D but is inhibited by cycloheximide. PKC-ϵ has some characteristics of a novel `early response' protein whose synthesis in newly passaged 3T6 cells is regulated at the translational level

Nonlinear stochastic systems : dynamic excitation of stayed masts by wind turbulence

Imperial Users onl

PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo

Epithelial differentiation including tight junction (TJ) formation occurs exclusively within the trophectoderm (TE) lineage of the mouse blastocyst. Here we examine mechanisms by which TJ protein membrane assembly might be regulated by protein kinase C (PKC) in the embryo. To overcome the inherent staging asynchrony of individual blastomeres within intact embryos, we have used isolated inner cell masses (ICMs) from early blastocysts to induce epithelial differentiation in their outer cells responding to their new cell contact pattern. Two TJ proteins examined retain their order of membrane assembly in isolated ICMs in culture as during normal development (early-assembling ZO-2 and late-assembling ZO-1{alpha}+), but this process is highly accelerated. Using six chemical modulators of PKC activity, we show here that PKC signalling is involved in the regulation of TJ membrane assembly. While indolactam-mediated PKC activation stimulates membrane assembly of both TJ proteins, TPA-mediated PKC activation stimulates only that of ZO-1{alpha}+. The PKC inhibitors Ro-31-8220, Ro-31-8425 and Gö 6983 suppress the stimulatory effect of both PKC activators on membrane assembly to varying extents according to inhibitor and TJ protein examined. Gö 6983 similarly inhibits ZO-2 and ZO-1{alpha}+ membrane assembly. PKC inhibition by Gö 6976 appeared to stimulate TJ membrane assembly. Despite the broad PKC isotype specificity of the inhibitors used, these data suggest that the two TJ proteins are differently regulated by PKC isotypes or subfamilies. As Gö 6983 uniquely affects aPKC (particularly PKC{zeta}) and we find that both PKC{delta} and {zeta} relocate upon activator treatment to colocalise partially with the TJ proteins in isolated ICMs, we suggest that at least PKC{delta} and {zeta} may play a central role in regulating TJ membrane assembly

Specific PKC isoforms regulate blastocoel formation during mouse preimplantation development preimplantation development

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by Na+/K+ ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four (y, u, L/E, ~) PKC isoforms and PKCA/PKD1 showed partial colocalisation with the tight junction marker ZO-1a+ in TE and all four PKCs (y, u, L/E, ~) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocystmorphogenesis. Specific inhibition of PKCy and ~ activity significantly delayed blastocyst formation. Although modulation of these PKCisoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, Na+/K+ ATPase a1 subunit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including Na+/K+ ATPase