Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells.

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP3), and on activation of protein kinase C (PKC) was studied in DDT1 MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A1 and some as yet unidentified P2Y receptor(s). 2. Activation of either receptor type stimulated the production of InsP3 and raised intracellular calcium in DDT1 MF2 cells. Similarly, the A1 selective agonist N6-cyclopentyladenosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP(4-14) and induced a PKC translocation to the plasma membrane as determined using [3H]-phorbol dibutyrate (PDBu) binding in DDT1 MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected gamma-PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of gamma-PKC-GFP and activation of PKC. 3. Doses of the A1 agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca2+ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 microM) and UTP (or ATP, 2.5 microM), which per se caused no detectable translocation of either gamma- or epsilon-PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A1 receptors synergistically increases Ca2+ transients and translocation of PKC in DDT1 MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.

Activation of protein kinase Czeta is essential for cytokine-induced metalloproteinase-1, -3, and -9 secretion from rabbit smooth muscle cells and inhibits proliferation.

Several matrix metalloproteinases (MMPs), including MMP-1, -3, and -9, mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP up-regulation by inflammatory cytokines involves interactions between several transcription factors, including activator protein-1 and nuclear factor kappaB (NF-kappaB). The upstream regulatory pathways are less well understood. We investigated the role of isoforms of protein kinase C (PKC) in basic fibroblast growth factor- and interleukin-1alpha-mediated MMP production from cultured rabbit aortic smooth muscle cells. A synthetic PKC inhibitor, RO318220, inhibited MMP-1, -3, and -9 production by 89 +/- 3, 75 +/- 18, and 89 +/- 9%, respectively. However, down-regulation of conventional and novel isoforms did not inhibit but rather increased MMP-9 production by 48 +/- 16%, implicating an atypical PKC isoform. Consistent with this, PKCzeta protein levels and activity were stimulated 3.3- and 13-fold, respectively, by basic fibroblast growth factor plus interleukin-1alpha and antisense oligonucleotides to PKCzeta significantly decreased MMP-9 formation by 62 +/- 18% compared with scrambled sequences. Moreover, adenovirus-mediated overexpression of a dominant-negative (DN) PKCzeta reduced MMP-1, -3, and -9 production by 78 +/- 9, 76 +/- 8, and 76 +/- 5%, respectively. DN-PKCzeta inhibited NF-kappaB DNA binding but did not affect ERK1/2 activation or AP-1 binding. Antisense PKCzeta oligonucleotides and DN-PKCzeta stimulated cell proliferation by 89 +/- 14% (n = 4) and 305 +/- 74% (n = 3), respectively (both p < 0.05). Our results show that PKCzeta is essential for cytokine-induced up-regulation of MMP-1, -3, and -9, most likely by activating NF-kappaB. Selective inhibition of PKCzeta is therefore a possible strategy to inhibit MMP production in inflammatory diseases such as atherosclerosis.