The role of cyclic-3′,5′-adenosine monophosphate in prostaglandin-mediated inhibition of basic calcium phosphate crystal-induced mitogenesis and collagenase induction in cultured human fibroblasts

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies characterised by synovial proliferation and non-inflammatory degradation of intra-articular collagenous structures. BCP crystals stimulate fibroblast and chondrocyte mitogenesis, metalloprotease secretion and prostaglandin production. As a tissue protective effect of prostaglandins has been suggested, we recently studied the effect of PGE₁ on BCP crystal-induced mitogenesis and collagenase mRNA accumulation in human fibroblasts (HF). We demonstrated a dose-dependent inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation. The mechanism of PGE₁ inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation was therefore explored. PGE₁ (100 ng/ml) increased HF intracellular cAMP 40-fold over control. BCP alone caused no such change but inhibited the PGE₁-induced increase in intracellular cAMP by at least 60%. The PGE₁-induced increase in intracellular cAMP was also blocked by the adenyl cyclase inhibitor, 2′,5′-dideoxyadenosine (ddA) (10 μM) and ddA reversed the PGE₁-mediated inhibition of BCP crystal-induced mitogenesis. Dibutyrul cAMP also inhibited BCP crystal-induced mitogenesis in a concentration-dependent manner. Agents which increase intracellular cAMP levels such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) mimicked the effect of PGE₁ on HF collagenase mRNA levels. PGE₁ inhibits the biologic effects of BCP crystals through the cAMP signal transduction pathway and such inhibition may have significant therapeutic implications.     

Molecular mechanism underlying adenosine receptor-mediated mitochondrial targeting of protein kinase C

Activation of protein kinase C (PKC) via adenosine receptors is known to be involved in the cardioprotection of ischemic preconditioning (IPC). Specifically, activation of PKCε is critical for cardioprotection. There is ample evidence that PKCε resides in cardiac mitochondria. However, the signals that promote translocation of PKCε are largely unknown. The present study was designed to determine whether and how adenosine receptor activation induces translocation of PKCε to mitochondria. Freshly isolated adult rat cardiac myocytes and rat heart-derived H9c2 were used in the study. Immunofluorescence imaging of isolated mitochondria showed that PKCε but not PKCδ was localized in mitochondria and this mitochondrial localization of PKCε was significantly increased by adenosine treatment. The adenosine-induced increase in PKCε-positive mitochondria was largely prevented not only by PKC inhibitor chelerythrine, but also by the HSP90 inhibitor geldanamycin and by siRNA targeting HSP90. Immunoblot analysis from percoll-purified mitochondria further demonstrated that adenosine mediated a significant increase in mitochondrial PKCε? but not PKCδ. This effect was blocked by inhibiting PKC activity with chelerythrine and bisindolylmaleimide. Furthermore, co-immunoprecipitation data showed that PKCε but not PKCδ was associated with TOM70 and HSP90, and this association was enhanced by adenosine treatment. Moreover, adenosine-induced association of PKCε with TOM70 was reduced by suppressing HSP90 expression with siRNA. In conclusion, we demonstrate that adenosine induces HSP90-dependent translocation of PKCε to mitochondria, possibly through mitochondrial import machinery TOM70. These results point out a novel mechanism in regulating PKC in mitochondria and suggest an important implication in ischemic preconditioning or postconditioning.     

Redistribution of protein kinase C during mitogenesis of human B lymphocytes

G0 human tonsillar B-lymphocytes were stimulated to divide by the polyclonal mitogen Staphylococcus Aureus Cowan strain 1 (SAC) and by the combined use of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the calcium ionophore ionomycin. The activities of protein kinase C, which requires Ca++ and phospholipid as co-factors, and a proteolytically cleaved form of this enzyme (protein kinase M), which is independent of calcium and phospholipid control, were determined in soluble and particulate fractions obtained from activated B cells. Treatment of G0 B cells with SAC or TPA together with ionomycin caused redistribution of protein kinase C from the soluble to the particulate fraction where the 80,000-Dalton protein kinase C was cleaved to give rise to a 50,000-Dalton form of the kinase which was also found in the cytoplasm. These data suggest that redistribution and proteolytic cleavage of protein kinase C are key signal transduction events in B cell mitogenesis.     

Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).     

Molecular mechanism of protein kinase C modulation of sodium channel alpha-subunits expressed in Xenopus oocytes.

The mechanism of modulation of sodium channel alpha-subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments.     

Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C.

The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C.     

Regulation of myeloid-specific calcium binding protein synthesis by cytosolic protein kinase C.

Two calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) is shown to enhance the inductive effect of RA and 1 alpha,25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1 alpha,25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1 alpha,25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.     

The mechanism of protein kinase C regulation

Protein kinase C (PKC) is a family of serine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis. Nine PKC genes have been identified in the human genome, which encode 10 proteins. Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations. Activation of PKC has been implicated in the regulation of cell growth and differentiation. This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.     

Molecular biology of protein kinase C signaling in cardiac myocytes.

The PKC family of serine/threonine kinases have been implicated in a diverse array of cellular responses. Adult cardiac myocytes express multiple PKC isozymes, which participate in the response of muscle cells to extracellular stimuli, modulate contractile properties, and promote cell growth and survival. Recently, the classification of this ubiquitous family of signaling molecules has been expanded from three to four subfamilies. This review will focus on the application of pharmacologic and molecular approaches to explore the biology of cardiac PKC isozymes. The availability of transgenic mice and peptide PKC modulators have been instrumental in identifying target substrates for activated cardiac PKC isozymes, as well as the identification of specific isozymes linked to distinct growth characteristics and cell phenotype. The rapid growth of knowledge in the area of PKC signaling and PKC substrate interactions, may result in the development of therapeutic modalities with the potential to arrest or reverse the progression of cardiovascular diseases.     

Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues.

Important determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II (CaMK-II), corresponding to residues 281-302 of the kinase alpha-subunit sequence, were identified. Replacement of Thr286 with Ala (CaMK-(281-302 Ala286)) had no effect on either the potency (IC50 = 2 MicroM) or inhibitory mechanism (competitive with ATP) using the catalytic fragment of CaMK-II. Single replacement of charged residues in CaMK-(281-302, Ala286) identified His282, Arg283, Lys291, Arg297, and Lys298 as important determinants (greater than 10-fold increase in IC50) for potent inhibition of CaMK-II. Glu285, Asp288, Lys291, Arg296, and Lys300 were not as essential (less than 4-fold change in IC50) for potent CaMK-II inhibition. Replacement of either Arg283, Lys291, or Arg297, and Lys298 with Ala did not alter the ATP-competitive mechanism of inhibition although the Ki values increased 16-530-fold. However, replacement of His282 with Ala decreased the IC50 by 20-fold and altered the mechanism of inhibition to noncompetitive with respect to ATP. The non-protonated form of His282 was functionally active since decreasing the pH from 7.5 to 5.5 increased the IC50 of CaMK-(281-302, Ala286) almost 20-fold. Histidine protonation also appeared to disrupt the autoinhibitory domain of intact forms of CaMK-II since preincubation of non-proteolyzed rat brain CaMK-II with calcium/calmodulin (in the absence of ATP) at pH 5.5 generated up to 16% calcium-independent activity when assayed at pH 5.5. Similarly, the level of calcium-independent activity of a baculovirus-expressed Asp286 mutant CaMK-II ((D286)mCaMK alpha) increased to almost 80% calcium independence when assayed at pH 5.5 compared to only 20% when assayed at pH 7.5. The levels of calcium-independent activity of both the (D286)mCaMK alpha (at pH 5.5 and 7.5) and the rat brain CaMK-II (at pH 5.5) were sensitive to the concentrations of both ATP and peptide substrate (syntide-2) in the assays. These data suggest that the basic residues Arg283, Lys291, Arg297, and Lys298 are important for potent inhibition of CaMK-II and that the non-protonated form of His282 may play a unique role in the ATP-directed mechanism of inhibition by the CaMK-II autoinhibitory domain.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

Role of protein kinase C in cellular regulation

Protein kinase C (PKC) consists of a family of closely related enzymes ubiquitously present in animal tissues. These enzymes respond to second messengers, Ca2+, diacylglycerol and arachidonic acid, to express their activities at membrane locations. Numerous hormones, neurotransmitters, growth factors and antigens are believed to transmit their signals by activation of a variety of phospholipases to generate these messengers. The various PKC isozymes, which exhibit distinct biochemical characteristics and unique cellular and subcellular localizations, may be differentially stimulated depending on the duration and strength of these messengers. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, secretion, gene expression, and neuronal plasticity and toxicity. The mechanisms of action of PKC in the regulation of these cellular functions are not entirely clear. Further study to identify the target substrates relevant to the various cellular functions is essential to define the functional diversity of this enzyme family.