Regular ArticleRegulation of Protein Kinase C in NG108-15 Cell Differentiation
Abstract
The involvement of PKC in NG108-15 cell differentiation was investigated. Differentiation with dBcAMP was associated with a decrease in total cellular phorbol ester binding. The histone-directed PKC activity was decreased in the soluble fraction. Northern and Western blotting revealed the presence of only PKC α but not PKC β and PKC γ among the calcium-dependent isoforms. Differentiation induced a decrease of cytosolic PKC α immunoreactivity, with no changes of mRNA content or appearance of PKC β and PKC γ isoforms. The low levels of PKCα in the soluble fraction suggest that the mRNA for this species is less efficiently translated in differentiated NG108-15 cells. The data suggest that downregulation of PKC α protein and kinase activity are associated with induction of neuronal morphology in NG108-15 cells.
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Selective activation of protein kinase C isoforms by angiotensin II in neuroblastoma X glioma cells
2004, Molecular and Cellular EndocrinologyDifferential activation of PKC isoforms by angiotensin II (AII) has been found in a variety of tissues in which this important octapeptide mediates its multitude of effects. To date, the PKC isoforms involved in mediating brain-specific effects are yet to be defined. In the present study, the identity of PKC isoforms coupled to AII stimulation was examined in the neuroblastoma X glioma hybrid cell line, NG108-15, by Western blot analysis. This cell line expresses both the AT1 and AT2 receptor subtypes, with the AT1 subtype predominating, and expression levels highly-upregulated when cells are in the differentiated state. Six PKC isoforms were examined in the present study, including three Ca2+ dependent (α, β, and γ), and three Ca2+ independent (δ, ε, and ζ) isoforms. NG108-15 cells were found to express PKC α, δ, ε, and ζ isoforms but not β or γ isoforms. Differential sensitivity of the PKC isoforms to AII stimulation was demonstrated, with AII causing a rapid and transient activation of the PKC α only in undifferentiated cells, whereas both PKC α and ε isoforms were responsive in differentiated cells. PKC activation was found to be both dose- and time-dependent. The data demonstrate the differential activation of PKC isoforms to AII stimulation in NG108-15 cells, with evidence supporting the involvement of the PKC α and ε isoforms in AII-mediated effects in the brain.
Protein kinase C anchoring deficit in postmortem brains of Alzheimer's disease patients
1999, Experimental NeurologyProtein kinase C (PKC) has been implicated in the pathophysiology of Alzheimer's disease (AD). The levels of particular isoforms and the activation of PKC are reduced in postmortem brain cortex of AD subjects. Receptors for activated C kinase (RACK) are a family of proteins involved in anchoring activated PKCs to relevant subcellular compartments. Recent evidence has indicated that the impaired activation (translocation) of PKC in the aging brain is associated with a deficit in RACK1, the most well-characterized member of this family. The present study was conducted to determine whether alterations in RACK1 occurred in cortical areas where an impaired translocation of PKC has been demonstrated in AD. Here we report the presence of RACK1 immunoreactivity in human brain frontal cortex for the first time and demonstrate a decrease in RACK1 content in cytosol and membrane extracts in AD when compared with non-AD controls. By comparison, the levels of the RACK1-related PKCβII were not modified in the same membrane extracts. These observations add a new perspective in understanding the disease-associated defective PKC signal transduction and indicate that a decrease in an anchoring protein for PKC is an additional determinant of this deficit.
Participation of protein kinase C α in 1,25-dihydroxy-vitamin D<inf>3</inf> regulation of chick myoblast proliferation and differentiation
1999, Molecular and Cellular EndocrinologyChanges in morphology and DNA synthesis in cultured myoblasts in response to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] have previously suggested that the vitamin D hormone may affect muscle cell proliferation and differentiation. However, this interpretation was not substantiated by measurement of specific biochemical markers of myogenesis. To study the effect of 1,25(OH)2D3 on muscle development, chicken embryo myoblasts were cultured for 1–6 days in the presence or absence of 1,25(OH)2D3 (10−9 M). The hormone increased DNA synthesis and decreased creatine kinase activity, indicating stimulation of cell proliferation and inhibition of myogenesis, in undifferentiated myoblasts (1 day of culture). At longer culture intervals, when myoblasts elongate and fuse to form differentiated myotubes, 1,25(OH)2D3 promoted myogenesis, as indicated by an inhibition of DNA synthesis and an increase in specific muscle differentiation markers as creatine kinase activity and myosin expression. The role of protein kinase C (PKC) in mediating the effects of hormone and the likely PKC isoform involved were also investigated. Increased PKC activity was observed during 1,25(OH)2D3 stimulation of myoblast proliferation whereas inhibition of PKC activity accompanied the effects of the hormone on myoblast differentiation. The specific PKC inhibitor calphostin suppressed hormone potentiation of DNA synthesis in proliferating myoblasts. 1,25(OH)2D3-dependent changes in the expression of PKC isoforms α, β, δ, ε and ζ during myogenesis were investigated by Western blot analysis. The early stimulation of myoblast proliferation by the hormone mainly correlated to increased PKC α expression whereas decreased PKC α levels were observed during the subsequent activation of myoblast differentiation. These results support that 1,25(OH)2D3 has a function in embryonic muscle growth and maturation, and PKC α may participate in the signal transduction pathway which mediates the response to the hormone.
Selective activation by bryostatin-1 demonstrates unique roles for PKCε in neurite extension and tau phosphorylation
1997, International Journal of Developmental NeurosciencePhorbol esters such as 12-O-tetradeonyl phorbol-13 acetate (TPA) induce a time-dependent biphasic effect on protein kinase C (PKC)-mediated events by fostering translocation of cytosolic (latent) PKC to the plasma membrane (where it is activated). Continued treatment, however, depletes the cell’s entire PKC complement and induces a functional stake of PKC inhibition. Previous studies from several laboratories have demonstrated that long-term TPA treatment, like treatment with PKC inhibitors, induces neuronal differentiation. Bryostatin-1 also induces translocation and overall downregulation of PKC following long-term treatment, yet, unlike TPA or PKC inhibitors, does not induce neuronal differentiation, promoting controversy regarding the role of PKC inhibition in neuronal differentiation. We demonstrate herein that, despite overall downregulation in human neuroblastoma cells, membrane-associated levels of one PKC isoform (PKCε) are actually increased following long-term bryostatin-1 treatment. Since previous studies have implicated this PKC isoform in phosphorylation of the microtubule-associated protein tau and in neuritogenesis, we examined the consequences of long-term bryostatin treatment on these phenomena. Treatment with 25 n-100 M bryostatin-1 for 72 h increased tau phosphorylation and inhibited neuritogenesis. By contrast, treatment with either TPA or the PKC inhibitor staurosporine did not induce tau phosphorylation and induced neurite elaboration. Bryostatin-1 antagonized neurite induction by staurosporine. These findings provide additional evidence for a unique role of PKCε in the regulation of tau phosphorylation and neuronal differentiation, and demonstrate that bryostatin-1 can function under certain conditions as a selective PKCε activator even following long-term treatment.
Protein F1/GAP-43 and PKC gene expression patterns in hippocampus are altered 1-2 h after LTP
1995, Molecular Brain ResearchThree days after long-term potentiation (LTP) there is a decrease in the gene expression of protein F1 (GAP-43) and γ-PKC in CA3 pyramidal cells that is correlated with the magnitude of LTP. We predicted these decreases would be preceded by an increment in gene expression. At 1 h, but not at 2 h after LTP, F1/GAP-43 and γ-PKC mRNA hybridization were increased, but increases were also observed after control stimulation. At both 1 and 2 h after LTP, changes in F1/GAP-43 hybridization were positively correlated with γ-PKC hybridization and negatively correlated with LTP magnitude. These data indicate that correlated alterations in F1/GAP-43 gene expression and synaptic efficacy can occur as early as 1 h after LTP and persist for days.
Several protein kinase C (PKC) isozymes were analyzed by immunoblot and immunocytochemistry in cultures of hippocampal neurones at several stages of differentiation. Our findings reveal the existence of two distinct patterns of expression. Firstly, conventional PKC isozymes α, β and γ, that are expressed at very low levels during the initial stages and then increase continuously with time of culture. Secondly, novel PKC isozymes δ, ϵ and ζ, whose contents increase very early to reach a maximum after three days of culture and then progressively decline. Specific proteolysis for PKC isozymes β and γ was observed throughout the period studied. The developmental profile obtained for the different PKC isozymes is discussed in relation to the differentiation of hippocampal neurones in culture.