Decrease In Protein Kinase C Research Articles

All-Trans Retinoic Acid Has a Potential Therapeutic Role for Diabetic Nephropathy.

PurposeThe aim of this study was to examine the effects of all-trans retinoic acid (ATRA) on diabetic nephropathy.Materials and MethodsWe measured amounts of urinary albumin excretion (UAE) after administrating ATRA to Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In order to understand the mechanism of action for ATRA, we administrated ATRA to examine its inhibitory action on the production of transforming growth factor-β1 (TGF-β1), protein kinase C (PKC), and reactive oxidative stress (ROS) in cultured rat mesangial cells (RMCs).ResultsAfter 16 weeks of treatment, UAE was lower in the ATRA-treated OLETF rats than in the non-treated OLETF rats (0.07±0.03 mg/mgCr vs. 0.17±0.15 mg/mgCr, p<0.01). After incubation of RMCs in media containing 30 or 5 mM of glucose, treatment with ATRA showed time- and dose-dependent decreases in TGF-β1 levels and ROS. Moreover, ATRA treatment showed a dose-dependent decrease in PKC expression.ConclusionATRA treatment suppressed UAE and TGF-β1 synthesis, which was mediated by significant reductions in PKC activity and ROS production. Our results suggest that ATRA has a potential therapeutic role for diabetic nephropathy.

In vivo and in vitro zinc (Zn) deficiency diminished T lymphocyte activity through the modulation of PKC isoenzymes. (87.46)

Abstract Zn is essential to numerous signaling proteins that share Zn-finger structures as a common motif. It is crucial for immunocompetent cells, as its deficiency leads to immune function alterations. The exact mechanisms involved in Zn regulation of lymphocyte activity are still unclear. Here we analyzed both the direct and the in vivo effect of Zn deficiency on normal T cells and the involvement of protein kinase C (PKC) (an enzyme with cysteine-rich domains) and its isoenzyme profile. For this purpose mitogen stimulated normal T cells were cultured in the absence or presence of specific intra- or extracellular Zn chelators. These effects were compared with those obtained in mitogen-stimulated lymphocytes purified from mice fed with Zn deficient diets. Proliferation, PKC activity and the pattern of PKC isoenzymes were evaluated. Zn chelators inhibited normal cell proliferation and their effects were reverted by in vitro Zn addition. Similar effects were observed in lymphocytes from Zn deficient mice. In both experimental conditions, a decrease in PKC activity and in mitogen-induced translocation of the enzyme to cell membranes were observed. These effects were accompanied by a decrease in PKC α and 𝛉 isoforms. These results show that Zn plays an important role in T cell growth and that its deficiency would alter the expression of key PKC isoenzymes that are essential signalling molecules involved in lymphocyte activation.

Overexpression of amyloid precursor protein reduces epsilon protein kinase C levels

Alzheimer’s disease (AD) is characterized by extracellular deposits of amyloid beta peptide (Aβ), a peptide that is generated upon proteolytic cleavage of amyloid precursor protein (APP). The events leading to the development of AD and their sequence are not yet fully understood. Protein kinase C (PKC) has been suggested to have a significant role in controlling neuronal degeneration and in the aberrant signal transduction taking place in AD. Several studies document a deficit in PKC levels and activity in brains of AD patients when compared with those of normal controls. Such a decrease in PKC could have serious implications since certain PKC isozymes were shown to drive the APP proteolytic cleavage into a non-amyloidogenic pathway. Reduced levels of distinct PKC isozymes could thus contribute to driving APP processing toward an amyloidogenic pathway. The direct cause for the down-regulation of PKC in AD brains is still unknown. In that respect, we tested in this study whether APP may play a role in PKC reduction. We show in three different cell lines (CHO, COS and BOSC) that overexpression of APP leads to decreased PKC levels. This decrease was found to be specific for the epsilon PKC isozyme whereas the levels of delta, alpha and conventional PKC remained unchanged. Furthermore, we observed this decrease for both active, membrane-associated and inactive, cytosolic epsilon PKC. APP-driven decrease in epsilon PKC is most likely mediated by a factor in the culture medium, since transfer of medium from cultured cells overexpressing APP to naïve, non-overexpressing cells, has also led to the selective decrease in epsilon PKC levels. Taken together, our results suggest that APP expression levels may play a role in the decrease of epsilon PKC levels in AD brains and could thus affect the responsiveness of AD brain tissues to growth factors and neurotransmitters.

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice.

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.

Transforming growth factor-β1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways

Previous studies have shown that transforming growth factor-β1 (TGF-β1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-β1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [ 35S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-β1 on matrix production. However, there was little, if any, effect on TGF-β1-dependent increases in [ 3H]thymidine incorporation, and TGF-β1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-β1 caused a dose-dependent increase in prostaglandin E 2 (PGE 2) production which was further increased by PKC inhibition. The increase was regulated by TGF-β1-dependent effects on phospholipase A 2 (PLA 2). Activation of PLA 2 inhibited TGF-β1 effects on PKC, and inhibition of PLA 2 activated TGF-β1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-β1-dependent increases in PKC. The effects of TGF-β1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed. These results support the hypothesis that TGF-β1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE 2 and protein kinase A (PKA). Inhibition of PKA also decreases TGF-β1-dependent proliferation. We have previously shown that PGE 2 stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways.

Regulation of protein kinase C δ by estrogen in the MCF-7 human breast cancer cell line

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) δ in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC δ isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC δ was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC δ expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC δ protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10 −10–10 −8 M estrogen for 7 days down-regulated specifically PKC δ mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC δ expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor β while the MCF-7 cells expressed predominantly estrogen receptor α and barely detectable levels of estrogen receptor β. These results suggest that the differential ability of estrogen to regulate PKC δ expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Antigen-mediated phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells: possible involvement of calcium/calmodulin

The differential implication of protein kinase C (PKC) isozymes in antigen- or PMA-induced phospholipase D (PLD) activation was investigated in rat basophilic leukemia (RBL-2H3) cells. In [ 3H]oleic acid-labeled cells, both antigen (100 ng/ml) and phorbol 12-myristate 13-acetate (PMA) (100 nM) produced a specific product of PLD activation, [ 3H]phosphatidylbutanol (Pbut) in the presence of butanol. Pretreatment of cells with a selective PKC inhibitor, Ro31–8425 (1–5 μM) inhibited PMA-stimulated PLD activity by 85%. In contrast, the antigen-stimulated PLD activity was much less sensitive to the inhibitor. RBL-2H3 cells express PKC α, β, δ, ε and ζ isozymes and down-regulation of PKC by exposure to PMA (20 nM) for 1–2 h caused rapid decrease in PKC α and β isozymes, leaving PKC δ, ε and ζ isozymes intact. Apparent decreases in the levels of PKC α and β to about 50% were observed after adding 20 nM PMA for 1 h, when PMA-stimulated PLD activity was inhibited by up to 70%. Decrease in antigen-stimulated PLD activity was evident after 2 h PMA-treatment, when PKC α and β decreased by nearly 70%. These results suggest that in the antigen-mediated PLD pathway PKC may be implicated but not play such a great role as PMA-stimulated pathway which is mediated through PKC α or β. Then, we have examined the involvement of calcium/calmodulin (CaM) in PLD activation by antigen, since the antigen-stimulated PLD activation showed the absolute requirement for extracellular calcium. Preincubation of RBL-2H3 cells with a CaM antagonist W-7 (20 μM) inhibited the antigen-stimulated PLD activity by 90%, but W-5, a chlorine-deficient analogue of W-7 that only weakly interact with CaM, caused little inhibitory effect. Another non-specific CaM antagonist, trifluoperazine (TFP) also inhibited PLD activation. These results suggest that calcium/CaM may be involved in the antigen-stimulated PLD activation.

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.