The effects on cellular proliferation and Ca2+ mobilization of analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP) and of agents that elevate the intracellular concentrations of cyclic nucleotides were compared in closely similar preparations of first-passage rabbit aortic vascular smooth-muscle cells. Proliferation induced by foetal-bovine serum was inhibited by 78% by 1 mM-8-bromo cAMP and by 42% by 1 mM-8-bromo cGMP. In the presence of 100 microM-isobutylmethylxanthine, 100 microM-forskolin increased intracellular cAMP concentration 5-fold and inhibited proliferation by 87%, but did not affect cGMP concentration or cell viability (ATP concentration). Similarly in the presence of 100 microM-isobutylmethylxanthine, 1 mM-SIN-1 (3-morpholinosydnonimine) elevated cGMP concentration 4-fold and inhibited proliferation by 48%, but did not affect cAMP or ATP concentration. Isobutylmethylxanthine (1 mM) elevated cAMP concentration by 3-fold and cGMP concentration by 20-fold and inhibited proliferation by 81%. Concentrations of 8-bromo cAMP, 8-bromo cGMP, forskolin or SIN-1 that inhibited proliferation did not affect the elevation of intracellular free Ca2+ concentration caused by 2% (v/v) foetal-bovine serum, 100 nM-5-hydroxytryptamine or 10 nM-angiotensin II. The results demonstrate that elevation of intracellular cAMP and cGMP concentrations both independently inhibit vascular smooth-muscle cell proliferation, but these effects on proliferation are not mediated by inhibition of Ca2+ mobilization.

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 B (NF-B). 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-1␣-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, PKC protein levels and activity were stimulated 3.3-and 13-fold, respectively, by basic fibroblast growth factor plus interleukin-1␣ and antisense oligonucleotides to PKC significantly decreased MMP-9 formation by 62 ؎ 18% compared with scrambled sequences. Moreover, adenovirus-mediated overexpression of a dominant-negative (DN) PKC reduced MMP-1, -3, and -9 production by 78 ؎ 9, 76 ؎ 8, and 76 ؎ 5%, respectively. DN-PKC inhibited NF-B DNA binding but did not affect ERK1/2 activation or AP-1 binding. Antisense PKC oligonucleotides and DN-PKC stimulated cell proliferation by 89 ؎ 14% (n ‫؍‬ 4) and 305 ؎ 74% (n ‫؍‬ 3), respectively (both p < 0.05). Our results show that PKC is essential for cytokine-induced up-regulation of MMP-1, -3, and -9, most likely by activating NF-B. Selective inhibition of PKC is therefore a possible strategy to inhibit MMP production in inflammatory diseases such as atherosclerosis. Remodeling of the extracellular matrix (ECM)1 occurs physiologically during morphogenesis, and pathologically in a variety of chronic inflammatory diseases, including atherosclerosis (1). Matrix degrading metalloproteinases (MMPs), which are key enzymes in extracellular matrix turnover at physiological pH, play a dual role in atherosclerosis. They are necessary for the smooth muscle cell migration and proliferation that underlie fibrous cap formation but also mediate the degradation of the ECM that underlies atherosclerotic plaque rupture and aneurysm formation (2). The MMP family of more than 20 proteases includes the gelatinases, collagenases, and stromelysins, which interact synergistically to degrade all the ECM components (3). The gelatinases comprise a 72-kDa gelatinase A (MMP-2), which tends to show constitutive pattern of regulation in connective tissue cells, and a 95-kDa gelatinase B (MMP-9) that is inducible by inflammatory cytokines, especially together with connective tissue growth factors. Both degrade the basement membrane around SMC, which permits SMC migration and proliferation (4 -6). On the other hand, the localization of stromelysin (MMP-3) and collagenase (MMP-1) in macrophage-rich shoulder regions of atheroscle...

Levels of PKC-alpha and delta expression appear to be indicative of response to anti-oestrogen therapy and could be useful in predicting a patient's suitability for endocrine therapy.

We analysed the expression of protein kinase C (PKC) isoforms in smooth muscle cells (SMC) in various states of differentiation, using Western blots and thermocycle amplification of mRNA. SMC isolated from intact tissues express three isoforms of PKC, namely a, 6, and c. Following cell culture, SMC additionally express mRNA for PKC-E, but significant amounts of the corresponding protein were not detected. Transformed SMC, such as the cell line DDT, MF-2, express both mRNA and protein for PKC-E, lack the 6 isoenyzme, whilst maintaining the expression of the a and < isoforms. Thus PKCd and E isoenzyme expression appears to vary with the state of differentiation of these cells, with PKC-E expression increasing as the cells become proliferative.