Selective post-transcriptional down-regulation of protein kinase C isoenzymes in leukemic T cells chronically treated with phorbol ester

Binding to, and activation of, protein kinase C (PKC) by phorbol ester (PE) tumor promoters may underlie their tumor-promoting activity. To study the effects of long-term PE treatment on regulation of cellular PKC, we adapted the human leukemic T cell line, Jurkat (JK), to continuous growth in the presence of PE. Such cells (JKPE) displayed loss of CD2 and CD3 cell surface molecules, known to play an important role in agonist-stimulated T cell activation, unresponsiveness to stimuli that induce interleukin 2 (IL2) receptor expression or IL2 production, change in the expression of several cell cycle-regulated genes, and a 6-fold reduction in cellular PKC enzymatic activity. This reduction was accompanied by the disappearance of a major approximately 82-kDa immunoreactive protein in JKPE cytosol when cell extracts were immunoblotted with a polyclonal anti-PKC peptide antibody cross-reactive with the PKC isoenzymes, alpha, beta, and gamma. Analysis with additional anti-peptide antibodies specific for alpha, beta, or gamma PKC indicated that all three types of PKC are expressed in JK cells; however, JKPE cells lost a major approximately 82 kDa immunoreactive cytosolic protein detectable with anti-PKC alpha antibody. In contrast, levels of expression and subcellular distribution of immunoreactive PKC beta and PKC gamma, as well as levels of mRNA specific for the three PKC isoenzymes, were not significantly affected by chronic PE treatment. These results indicate that PE-mediated reduction of PKC in JKPE cells is selective and occurs at the protein, not mRNA, level, and support the notion that distinct isoenzymes encoded by the PKC multigene family may be independently regulated. Moreover, the correlation between phenotypic and functional changes on one hand, and the selective reduction of PKC alpha on the other, raises the possibility that expression of CD2 and/or CD3 and functional activation in JKPE cells are preferentially regulated by PKC alpha.     

Protein kinase C (PKC) alpha and PKC theta are the major PKC isotypes involved in TCR down-regulation

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCalpha and PKCtheta were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser(126) and the di-leucine-based receptor-sorting motif of the CD3gamma chain. Finally, we found that PKCtheta was mainly implicated in down-regulation of directly engaged TCR, whereas PKCalpha was involved in down-regulation of nonengaged TCR.     

Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation

PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131-139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant alpha, beta(1), beta(2), gamma, delta, zeta and eta PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of alpha, beta(1) and beta(2) PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.     

The presence in a mouse T cell line of a 97-kDa protein kinase C (PKC) with characteristics similar to known members of the novel PKC subgroup and its possible role in lymphocyte gene expression.

The receptor for tumor-promoting phorbol esters has been shown to be the Ca+2/phospholipid dependent enzyme protein kinase C (PKC). There are two major groups of PKC, the conventional PKC isotypes alpha, beta I, beta II, gamma) and the novel Ca+2-independent PKC (delta, epsilon, zeta, eta). Phorbol esters previously have been demonstrated to increase human IFN-gamma gene expression after treatment of a murine T cell line (Cl 9) that has been transfected with human IFN-gamma genomic DNA. In contrast, treatment with Ca+2 ionophore alone or in combination with phorbol ester did not enhance IFN-gamma production in a synergistic manner above the level obtained with phorbol ester treatment alone. To determine whether the lack of effect of Ca+2 ionophore is due to a defect in PKC, we compared the level of PKC autophosphorylation in the mouse T cell line (Cl 9), a mouse epidermal cell line (JB6), and purified rat brain PKC by in vitro kinase assays. The results demonstrate that instead of the expected 80-kDa autophosphorylated PKC band seen in purified rat brain PKC or mouse JB6 cell lysates, only a novel 97-kDa Ca+2-independent phosphoprotein was observed in Cl 9 cells. To ascertain if there was any nucleic acid sequence similarity to PKC epsilon, we hybridized Cl 9 poly(A+) RNA with a cloned fragment of the PKC epsilon gene and observed two hybridizing RNA bands (4.4 and 4.0 kb). Our results suggest that the 97-kDa phosphoprotein is similar to, but not identical with, PKC epsilon and is the major PKC expressed in the Cl 9 murine T cell line. These data suggested that the 97-kDa PKC may be responsible for the induction of both the transfected human IFN-gamma gene and the endogenous murine IL-2R alpha-chain.     

gamma delta T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium

We have investigated the expression of the alpha beta and the gamma delta T cell receptor (TCR) in the human intestine. By immunohistology we found that 39% of CD3+ intraepithelial lymphocytes (IEL) expressed the gamma delta TCR compared to 3% of CD3+ lamina propria lymphocytes (LPL). Cytofluorometric analysis of isolated cells revealed a significantly higher proportion of gamma delta T cells among CD3+ IEL compared to LPL and peripheral blood lymphocytes. This was due to an increase in both CD8+ (low density) and CD4-CD8- gamma delta T cells in IEL. Most alpha beta IEL expressed high-density CD8. About 30% of both IEL and LPL expressed CD25 (alpha-chain of the IL-2 receptor). HML-1 expression was detected on nearly all IEL and on 27% of LPL. CD25 and HML-1 were preferentially expressed on intestinal alpha beta and gamma delta T cells, respectively. Thus, human gamma delta T cells are located preferentially in the gut epithelium and are phenotypically different from alpha beta T cells, which constitute the majority of both LPL and IEL.     

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.     

Protein kinase C gamma (PKC gamma): function of neuron specific isotype

The gamma isotype of protein kinase C (PKC gamma) is a member of the classical PKC (cPKC) subfamily which is activated by Ca(2+) and diacylglycerol in the presence of phosphatidylserine. Physiologically, PKC gamma is activated by a mechanism coupled with receptor-mediated breakdown of inositol phospholipid as other cPKC isotypes such as PKC alpha and PKC beta. PKC gamma is expressed solely in the brain and spinal cord and its localization is restricted to neurons, while PKC alpha and PKC beta are expressed in many tissues in addition to the brain. Within the brain, PKC gamma is the most abundant in the cerebellum, hippocampus and cerebral cortex, where the existence of neuronal plasticity has been demonstrated. Pharmacological and electrophysiological studies have shown that several neuronal functions, including long term potentiation (LTP) and long term depression (LTD), specifically require PKC gamma. Generation of mice deficient in PKC gamma provided more information regarding the physiological functions of this isotype. PKC gamma deficient mice (i) have modified long term potentiation (LTP) in hippocampus, (ii) exhibit mild deficits in spatial and contextual learning (iii) exhibit impaired motor coordination due to persistent multiple innervations of climbing fibers on Purkinje cells, (iv) show attenuation of opioid receptor activation, and (v) show decreased effects of ethanol on type A of gamma-aminobutyric acid (GABA) receptor. Furthermore, a point mutation in the PKC gamma gene may contribute to retinitis pigmentosa and Parkinsonian syndrome. This article reviews the specific functions of this neuron-specific isotype of PKC in neuronal signal transduction.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

Retinoic acid-specific induction of a protein kinase C isoform during differentiation of HL-60 cells.

Human promyelocytic leukemia cells (HL-60) were treated with several differentiation inducers, then the changes in the activity of cytosolic protein kinase C (PKC) isoforms were examined by hydroxylapatite chromatography and the species of the isoforms were determined immunologically. In three undifferentiated HL-60 cell lines examined, PKC alpha and beta isoforms were present, but PKC gamma isoform was not detected. When the cells were induced by dimethylsulfoxide, dibutyryl cAMP, or nicotinamide to differentiate into granulocytes, these two PKC isoforms each increased to about 2- to 3-fold. When retinoic acid was used as the inducer, in addition to PKC alpha and beta, a third PKC isoform appeared. This isoform was clearly distinct from rat PKC alpha, beta, and gamma, immunologically. This isoform showed a distinctly lower Ca(2+)-requirement (3 microM) than that of PKC alpha or beta (100 microM) and was more dependent on cardiolipin and phosphatidylethanolamine, compared with PKC alpha, beta, and gamma. These results suggest that while the increases in the activities of PKC alpha and beta isoforms are common in the differentiation program initiated by several inducers, including retinoic acid, the emergence of an unclassified PKC isoform is a retinoic acid-specific process.     

High expression of the gamma5 isoform of G protein in neuroepithelial cells and its replacement of the gamma2 isoform during neuronal differentiation in the rat brain

High concentrations of G proteins, which include multiple isoforms of each subunit, alpha, beta, and gamma, are expressed in the adult brain. In this study, we concentrated attention on changes of these isoforms during embryonic development in the rat brain. Concentrations of gamma2 as well as GoAalpha, GoBalpha, and beta2 were low in early embryogenesis and then increased, whereas expression of gamma5, in contrast, was initially high followed by a drop, with only very low levels observed throughout postnatal development. Among the other isoforms, Gi1alpha, G(s)alpha-short, G12alpha, G13alpha, beta4, gamma3, gamma7, and gamma12 were present in the embryonic brain at low levels, but their levels markedly increased after birth. In contrast, the levels of Gi2alpha, G(s)alpha-long, Gq/11alpha, and beta1 were essentially constant throughout. Immunohistochemical staining of the brain vesicles in the embryos showed gamma5 to be specifically expressed in the proliferative region of the ventricular zone, whereas gamma2 was mainly present in differentiated neuronal cells of the marginal zone. Furthermore, differentiation of P19 mouse embryonal carcinoma cells to neuronal cells with retinoic acid induced the expression of gamma2 and a decrease of gamma5, the major isoform in the undifferentiated state. These results suggest that neuronal differentiation is responsible for the on/off switch of the expression of gamma2 and gamma5 subunits.     

Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.     

Cell surface stability of gamma-aminobutyric acid type A receptors. Dependence on protein kinase C activity and subunit composition

Type A gamma-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of alpha, beta, and gamma subunits. Although cell surface expression is essential for GABA(A) receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant alpha(1)beta(2) or alpha(1)beta(2)gamma(2) receptors was analyzed in human embryonic kidney cells. Alpha(1)beta(2)gamma(2) but not alpha(1)beta(2) receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABA(A) receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABA(A) receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the gamma(2) subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block alpha(1)beta(2)gamma(2) receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block alpha(1)beta(2)gamma(2) receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of gamma(2)-containing receptors. Together, our results demonstrate that the surface stability of GABA(A) receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.     

Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.     

Functional properties of recombinant rat GABAA receptors depend upon subunit composition.

GABA-gated chloride channels were expressed in human embryonic kidney cells following transfection of cDNAs encoding the alpha 1, beta 2, and gamma 2 subunits of the rat GABAA receptor (GABAR). Functional properties were determined using patch-clamp techniques in the whole-cell and outside-out configurations. Large whole-cell currents were observed in cells expressing the alpha 1 beta 2, alpha 1 gamma 2, and alpha 1 beta 2 gamma 2 subunit combinations. The unique characteristics of GABAR channels consisting of these subunit combinations depended upon the presence or absence of beta 2 and gamma 3 subunits. GABA-activated currents in cells expressing GABARs with the beta 2 subunit desensitized faster and showed greater outward rectification, and the channels had a shorter mean open time than GABARs composed of alpha 1 gamma 2 subunits. When the gamma 2 subunit was present the resulting GABAR channels had a larger conductance. The slope of the concentration-response curve was significantly steeper for GABARs composed of alpha 1 beta 2 gamma 2 subunits compared with GABARs consisting of alpha 1 beta 2 or alpha 1 gamma 2 subunit combinations.