Protein kinase C isozymes in human megakaryoblastic leukaemia cell line, MEG-01: possible involvement of the isozymes in the differentiation process of MEG-01 cells

The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG-01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG-01 expressed mRNAs of PKCα, -βI, -βII, -δ, -ε, -η, -θ and -ζ, but not PKCγ. At the protein molecule level, MEG-01 was observed to express PKCα, -βI, -βII, -ε, -θ and -ζ, but lack -γ, -δ and -η. When differentiation of MEG-01 was induced by 100 n M 12-O-tetradecanoyl-phorbol-13-acetate (TPA), rapid translocation from cytosol to membrane fraction and down-regulation of PKCα, -ε and -θ was observed in 1–2 h. On the other hand, PKCβI and -βII were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down-regulation, especially βII, was very slow. The myristoylated, alanine-rich C kinase substrate (MARCKS) in the membrane fraction of MEG-01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time-course study of TPA treatment indicated that incubation of the cells for 30 min is sufficient for differentiation. These results strongly suggest that the activation of PKCα, -ε and -θ is involved in the initiation of differentiation, and that PKCβI and -βII have important roles in the maintenance of differentiation. Although PKCζ was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.     

Protein kinase C isoforms α, δ and θ require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells)

Summary Protein kinase C (PKC) isoforms are potentially important as modulators of the insulin signalling chain and could be involved in the pathogenesis of cellular insulin resistance. We have previously shown that phorbol ester stimulated PKC β1 and β2 as well as tumor necrosis factor-α (TNFα) stimulated PKC ɛ inhibit human insulin receptor (HIR) signalling. There is increasing evidence that the insulin receptor substrate-1 (IRS-1) is involved in inhibitory signals in insulin receptor function. The aim of the present study was to elucidate the role of IRS-1 in the inhibitory effects of protein kinase C on human insulin receptor function. HIR, PKC isoforms (α, β1, β2, γ, δ, ɛ, η, θ and ζ) and IRS-1 were coexpressed in human embryonic kidney (HEK) 293 cells. PKCs were activated by preincubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (CTPA) (10^––7 mol/l) following insulin stimulation. While PKCs α, δ and θ were not inhibitory in HEK 293 cells which were transfected only with HIR and PKC, additional transfection of IRS-1 induced a strong inhibitory effect of these PKC isoforms being maximal for PKC θ (99 ± 1.8 % inhibition of insulin stimulated receptor autophosphorylation, n = 7, p < 0.001). No effect was seen with PKC γ, ɛ, ζ and η while the earlier observed insulin receptor kinase inhibition of PKC β2 was further augmented (91 ± 13 %, n = 7, p < 0.001 instead of 45 % without IRS-1). The strong inhibitory effect of PKC θ is accompanied by a molecular weight shift of IRS-1 (183 kDa vs 180 kDa) in the sodium dodecyl sulphate polyacrylamide gel. This can be reversed by alkaline phosphatase treatment of IRS-1 suggesting that this molecular weight shift is due to an increased phosphorylation of IRS-1 on serine or threonine residues. In summary, these data show that IRS-1 is involved in the inhibitory effect of the PKC isoforms α, β2, δ and θ and it is likely that this involves serine/threonine phosphorylation of IRS-1. [Diabetologia (1998) 41: 833–838] Received: 11 February 1998 and in revised form 2 April 1998     

PMA-induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level

We investigated whether members of the protein kinase C (PKC) family of enzymes could play a role in the nuclear events involved in megakaryocytic differentiation. PKC activity was analysed using a serine substituted specific peptide, which enabled us to evaluate the whole catalytic activity in the pluripotent haemopoietic HEL cell line treated with 10⁻⁷  m phorbol myristate acetate (PMA) or haemin. In parallel, the subcellular distribution of different PKC isoforms (α, βI, βII, γ, δ, ε, θ, η, ζ) was evaluated by Western blot. PKC catalytic activity in the nuclei of HEL cells showed a peak after acute (30 min) treatment with PMA, followed by a significant ( P  < 0.05) decline after prolonged exposure (72 h) to the same agonist, when most HEL cells had acquired a differentiated megakaryocytic phenotype. Western blot analysis of nuclear lysates consistently showed a significant increase of PKC-α, -βI, -ε, -θ and -ζ isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC-ζ isoforms. Moreover, PKC-δ appeared in HEL nuclei only after 72 h of exposure to PMA. On the other hand, neither the catalytic activity nor the immunoreactivity of the different PKC isoforms showed remarkable variations in nuclei of HEL cells induced to differentiate along the erythroid lineage with 10⁻⁷  m haemin.
The possible implications of these findings for a better understanding of the molecular events underlying the process of megakaryocytic differentiation are discussed.     

A case of μ heavy-chain disease associated with hyperglobulinemia, anemia, and a positive Coombs test

 We report here for the first time a patient with μ heavy-chain disease (HCD), hyperimmunoglobulinemia, and a positive direct antiglobulin test (DAT, Coombs test). The heavy-chain diseases involve the proliferation of lymphoplasma cells of B cell origin and are characterized by the production of incomplete heavy chains devoid of light chains. The association of μ heavy-chain disease with either hyperglobulinemia or a positive DAT has not been reported in the literature to date. In this patient, immunofixation of serum proteins with monospecific antisera to α-, γ-, μ,- or δ-chains and to κ- and λ-chains revealed a precipitation band with antibody to IgM, but not with κ and λ light-chain antibodies, indicating μ heavy-chain disease. Hyperglobulinemia was present, which is very uncommon for HCD. A DAT of the patient's red blood cells (RBC) was found to be strongly positive for anti-IgG but negative for anti-IgM, -IgA, -C3c, and -C3d. However, when the eluate from the patient's red blood cells was investigated with nephelometry, it was found to contain antigens reactive with anti-γ as well with anti-μ-antiserum. When a DAT was performed with a randomly chosen test cell incubated with the eluate, the antibody-containing eluate was shown to react with anti-IgG as well as with anti-IgM-antiserum. In summary, the eluate from the patient's RBCs contained IgG and an immunoglobulin structure reactive with anti-IgM in an RBC agglutination assay as well as with anti-μ antiserum in a nephelometric investigation. Whether this IgM on the patient's erythrocytes is penta- or oligomeric, complete IgM, or the heavy chain cannot be concluded from these observations. Received: May 15, 1998 / Accepted: August 24, 1998     

Protein kinase C expression in salivary gland acinar epithelial cells in non-obese diabetic mice,an experimental model for Sjögren''s syndrome

We planned to investigate the expression of protein kinase C (PKC) isoforms in acinar epithelial cells of salivary glands in the non-obese diabetic (NOD) mouse to find out if they develop changes of the PKC system like those seen in the human counterpart, i.e. in Sjögren's syndrome. Parotid, submandibular, and sublingual glands from NOD and control BALB/c mice were stained with a panel of monoclonal antibodies directed against conventional (, , and ), novel (, , and ), and atypical ( and ) PKC isoforms using the streptavidin/HRP method. Similarly to human labial salivary glands, acinar epithelial cells of the healthy control BALB/c mice contained two of the conventional PKC isoforms, and . Acinar and ductal epithelial cells also contained the atypical PKC isoforms and . PKC isoforms , , , and were not found. NOD mice which displayed focal sialadenitis contained the same conventional and atypical PKC isoforms. The acinar cells in NOD mice, in contrast to the Sjögren's syndrome patients, did not lack PKC or . On the contrary, PKC and staining was stronger than in the control BALB/c mice. The present results demonstrate that both conventional and atypical PKC isoforms participate in the salivary epithelial cell biology and that there are mouse strain-associated and/or disease state-associated changes in their expression. The lack of PKC and isoforms found in Sjögren's syndrome was not reproduced in NOD mice, which discloses one more difference between the human disease and its NOD mouse model.     

Protein kinase C isoforms β 1 and β 2 inhibit the tyrosine kinase activity of the insulin receptor

Summary Downregulation of insulin receptor tyrosine kinase (IRK) activity yields to impaired insulin signalling and contributes to the pathogenesis of cellular insulin resistance. Activation of protein kinase C (PKC) by different agents is associated with an inhibition of IRK activity in various cell types. There is evidence that this effect on IRK activity might be mediated through phosphorylation of specific serine residues of the insulin receptor β -subunit. Neither the domains of the IRK where inhibiting serine phosphorylation occurs nor the PKC isoform responsible for IRK inhibition have been identified. PKC consists of a family of at least 12 isoforms. The aim of the present study was to determine which PKC isoform might be capable of IRK inhibition. The human insulin receptor and the PKC isoforms α, β 1, β 2, γ , δ , ɛ , η , θ and ζ were overexpressed in human embryo kidney fibroblasts (HEK 293 cells) in order to answer this question. PKCs were activated by preincubation with the phorbolester (TPA) (10⁻⁷ mol/l) following insulin stimulation of the cells. When the IRK was coexpressed with the PKC isoforms β 1 and β 2, a 50 ± 15.7 and 45 ± 10.1 % inhibition of tyrosine autophosphorylation of IRK was observed while coexpression with the other isoforms did not significantly modify IRK autophosphorylation. The data suggest that the PKC isoforms β 1 and β 2 might be candidates for insulin receptor inhibition. [Diabetologia (1997) 40: 863–866] Received: 3 March 1997 and in revised form: 17 April 1997     

The expression of the p85α subunit of phosphatidylinositol 3-Kinase is induced by activation of the peroxisome proliferator-activated receptor γ in human adipocytes

Abstract Aims/hypothesis. Thiazolidinediones are new oral antidiabetic drugs that activate the nuclear receptor PPARγ. Our aim was to identify potential target genes of PPARγ in the human adipocyte in order to clarify how thiazolidinediones improve insulin sensitivity. Methods. The effect of BRL 49 653 (Rosiglitazone) on the mRNA expression of insulin receptor, insulin receptor substrate-1, p85α, p110α and p110β subunits of phosphatidylinositol 3-kinase, Glut 4 and hormone sensitive lipase was examined in isolated adipocytes. Target mRNA levels were determined by RT-competitive PCR. Results. The BRL 49 653 (1 μmol/l) increased the mRNA concentrations of p85αPI-3 K (264 ± 46 vs 161 ± 31 amol/μg total RNA, p = 0.003) whithout affecting the expression of the other mRNAs of interest. This effect was dose-dependent (K_0.5 = 5 nmol/l) and was reproduced by a specific activator of RXR, indicating that it was probably mediated by the PPARγ/RXR heterodimer. The BRL 49 653 also increased the amount of p85αPI-3K protein in adipose tissue explants (71 ± 19 %). In addition, BRL 49 653 produced a more than twofold increase in insulin stimulation of phosphatidylinositol 3-kinase activity and significantly enhanced the antilipolytic action of insulin. Conclusion/interpretation. This work demonstrates that the gene of p85αPI-3K is probably a target of PPARγ and that thiazolidinediones can improve insulin action in normal human adipocytes. Although the precise mechanism of action of BRL 49 653 on PI3-Kinase activity is not completely clear, these findings improve our understanding of the insulin-sensitizing effects of the thiazolidinediones, possible drugs for the treatment of Type II (non-insulin-dependent) diabetes mellitus. [Diabetologia (2001) 44: 544–554] Received: 18 May 2000 and in revised form: 2 January 2001     

Effect of PKC Isozyme Inhibition on Forskolin-Induced Activation of BKCa Channels in Rat Pulmonary Arterial Smooth Muscle

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BK_Ca) channels in vascular smooth muscle and cause vasodilatation. In pulmonary vascular smooth muscle (PVSM), BK_Ca channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BK_Ca channel causes pulmonary vasoconstriction. Protein kinase C (PKC) modulates BK_Ca channels in systemic vascular smooth muscle, but little is known about the effect of PKC on BK_Ca channel activity in PVSM. A novel finding from our laboratory showed that PKC activates BK_Ca channels in rat pulmonary arterial smooth muscle and, having observed that cAMP-elevating agents also open BK_Ca channels, we hypothesized that PKC may open BK_Ca channels via a cAMP-dependent mechanism. Forskolin (10 μM), an activator of adenylyl cyclase, which increases cAMP concentration, opened BK_Ca channels in single pulmonary arterial smooth muscle cells (PASMC) of the Sprague–Dawley rat. The effect of forskolin was completely blocked by the PKC inhibitor Go 6983, which selectively blocks the α, β, δ, γ, and ζ PKC isozymes, and, by rottlerin, which selectively inhibits PKCδ, and partially blocked by Go 6976, which selectively inhibits PKCα PKCβ, and PKCμ. These results indicate that specific PKC isozymes mediate forskolin-induced activation of BK_Ca channels in PASMC, which suggests that a signaling pathway involving PKC activation and cAMP exists in pulmonary arterial smooth muscle to open BK_Ca channels.