Elina Ekokoski

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL‐5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of 3H‐thymidine in a dose‐ and time‐dependent manner in the cells. At least a 24‐h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of 3H‐thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH‐mediated incorporation of 3H‐thymidine. The effect of ATP was apparently mediated via a G‐protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL‐5 cells. In addition, ATP stimulated the expression of a 62 kDa c‐fos dependent protein in a dose‐ and time‐dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c‐fos.

Design, Synthesis, and Biological Activity of Isophthalic Acid Derivatives Targeted to the C1 Domain of Protein Kinase C

Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([(3)H]PDBu) from PKC alpha and delta at K(i) values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time, that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.

Mechanisms of P2 receptor-evoked DNA synthesis in thyroid FRTL-5 cells.

The expression of the P2 receptors and their functional responses were studied in rat thyroid FRTL‐5 cells. RT‐PCR analysis revealed transcripts for the G protein‐coupled P2Y2, P2Y4 and P2Y6 receptors, and for the transmitter‐gated ion channel P2X3, P2X4 and P2X5 subunits. In Fura‐2‐loaded cells, UTP, ATP, ATPγS or UDP increased [Ca2+]i, and behaved as potent full agonists, while 2‐Methylthio‐ATP (2‐MeSATP), α,β‐methylene‐ATP (α,β‐meATP) and pure ADP were weak agonists. The agonist‐mediated [Ca2+ ]i increases were diminished in Ca2+ ‐free buffer, and by pertussis toxin (PTX) or suramin treatments. ATP, UTP, UDP and ATPγS increased 3H‐thymidine incorporation into DNA and expression of the protooncogenes c‐Fos and c‐Jun, while 2‐MeSATP was ineffective, and α,β‐meATP gave a response only at 100‐μM dose. The ATP‐stimulated expression of c‐Fos and c‐Jun was dependent on Ca2+, and protein kinase C, but not on calmodulin or Ca2+/calmodulin‐dependent protein kinase II. Extracellular signal‐regulated kinases (ERK1 and ERK2) are also involved as the MEK inhibitor, PD98059, reduced both ATP‐evoked 3H‐thymidine incorporation and c‐Fos and c‐Jun expression. These results indicate that multiple P2Y receptor subtypes and at least the P2X5 subtype are functionally expressed in FRTL‐5 cells, and that nucleotides acting via P2 receptors are involved in the regulation of DNA‐synthesis.

Current Status and Future Prospects of C1 Domain Ligands as Drug Candidates

The second messenger diacylglycerol (DAG) plays a central role in the signal transduction of G-protein coupled receptors and receptor tyrosine kinases by binding to C1 domain of effector proteins. C1 domain was first identified in protein kinase C (PKC) which comprises a family of ten isoforms that play roles in diverse cellular processes such as proliferation, apoptosis and differentiation. Aberrant signaling through PKC isoforms and other C1 domain-containing proteins has been implicated in several pathological disorders. Drug discovery concerning C1 domains has exploited both natural products and rationally designed compounds. Currently, molecules from several classes of C1 domain-binding compounds are in clinical trials; however, still more have the potential to enter the drug development pipeline. This review gives a summary of the recent developments in C1 domain-binding compounds.

The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment.

C1 Domain-Targeted Isophthalate Derivatives Induce Cell Elongation and Cell Cycle Arrest in HeLa Cells

Diacylglycerol (DAG)-mediated signaling pathways, such as those mediated by protein kinase C (PKC), are central in regulating cell proliferation and apoptosis. DAG-responsive C1 domains are therefore considered attractive drug targets. Our group has designed a novel class of compounds targeted to the DAG binding site within the C1 domain of PKC. We have previously shown that these 5-(hydroxymethyl)isophthalates modulate PKC activation in living cells. In this study we investigated their effects on HeLa human cervical cancer cell viability and proliferation by using standard cytotoxicity tests and an automated imaging platform with machine vision technology. Cellular effects and their mechanisms were further characterized with the most potent compound, HMI-1a3. Isophthalate derivatives with high affinity to the PKC C1 domain exhibited antiproliferative and non-necrotic cytotoxic effects on HeLa cells. The anti-proliferative effect was irreversible and accompanied by cell elongation. HMI-1a3 induced down-regulation of retinoblastoma protein and cyclins A, B1, D1, and E. Effects of isophthalates on cell morphology, cell proliferation and expression of cell cycle-related proteins were different from those induced by phorbol 12-myristate-13-acetate (PMA) or bryostatin 1, but correlated closely to binding affinities. Therefore, the results strongly indicate that the effect is C1 domain-mediated.

HIV-1 Tat-peptide inhibits protein kinase C and protein kinase A through substrate competition.

HIV-1 Tat-peptide is widely used as a vector for cargo delivery into intact cells. As a cationic, arginine-rich peptide it can readily penetrate the plasma membrane and facilitate the penetration of impermeable bioactive molecules such as proteins, peptides, nucleic acids and drugs. Although at first considered as an inert vector, recent studies have however shown that it might have effects on its own on various cellular processes. In the present study we have investigated the effects of the Tat-peptide(48-60) on two basic serine/threonine kinases, protein kinase C and A, since earlier studies have shown that certain arginine-rich peptides or proteins might have a modulatory effect on their activity. In in vitro studies, Tat-peptide inhibited PKC alpha in a concentration-dependent manner with an IC(50)-value of 22nM and PKA with an IC(50)-value of 1.2 microM. The mode of inhibition was studied in the presence of increasing concentrations of a substrate peptide or ATP. Tat-peptide competed with the kinase substrates, however it did not compete with ATP. In a panel of 70 kinases Tat-peptide showed inhibitory activity at least towards other AGC-family kinases (PKB, SGK1, S6K1, MSK1), CAMK-family kinases (CAMK1 and MELK) and a STE family kinase (MKK1). In HeLa cells Tat-peptide inhibited the phorbol ester-evoked ERK1/2 phosphorylation suggesting that Tat inhibited PKC also in intact cells. In thyroid cells Tat-peptide attenuated sphingosylphosphorylcholine-evoked Ca(2+)-fluxes, which have earlier been shown to be dependent on PKC. Taken together, these results indicate that the Tat-peptide(48-60) is a potent inhibitor which binds to the substrate binding site of the basophilic kinase domain.

Extracellular ATP-mediated phospholipase a2 activation in rat thyroid FRTL-5 cells: Regulation by a Gi/Go protein, Ca2+, and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A2 (PLA2) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL‐5 cells. The PLA2 activity was determined by measuring the release of [3H]‐arachidonic acid (AA) from prelabeled cells. ATP evoked a dose‐ and time‐dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a Gi/Go protein. The AA release was also diminished by chelating extracellular Ca2+ with EGTA or by inhibiting influx of Ca2+ using Ni2+. Although the activation of protein kinase C (PKC) by 12‐phorbol 13‐myristate acetate (PMA) alone did not induce any AA release, the ATP‐evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP‐evoked AA release and the mitogen‐activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP‐evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC‐mediated activation of MAP kinase. Inhibiting Src‐like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA2 activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP‐evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA2 by a Gi/Go protein‐dependent mechanism. Moreover, Ca2+, PKC, MAP kinase, and Src‐like kinases are also involved in this regulatory process. J. Cell. Physiol. 183:155–162, 2000.

The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC(50) values: 212-310nM), whereas E6 and G8 inhibited PKCɛ activity (IC(50) values: 103-233nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.

Effects of 5,8,11,14-eicosatetraynoic acid on thapsigargin-induced calcium entry, and intracellular pH in thyroid FRTL-5 cells

The effect of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of lipoxygenase and cytochrome P-450 epoxygenase enzymes, on calcium fluxes was investigated in Fura 2 loaded rat thyroid FRTL-5 cells. ETYA per se released sequestered calcium. ETYA also inhibited calcium influx in thapsigargin-stimulated cells in dose-dependent manner. Addition of calcium to cells treated with ETYA and stimulated with thapsigargin in a calcium-free buffer resulted in a blunted increase in intracellular free calcium compared with the response in control cells. In addition, ETYA per se acidified the cytosol in a dose-dependent manner. Acidification of the cytosol with the K+/H+ ionophore nigericin also decreased thapsigargin-induced calcium entry, but not to the same extent as that seen in cells treated with ETYA. The results suggest that ETYA is a potent modulator of calcium entry, and that part of the inhibitory effect of ETYA may be due to the ETYA-induced acidification of the cytosol.