Alton L. Boynton

Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases.

Recent studies suggest that the ability to inhibit the activity of certain serine/threonine protein phosphatases underlies the toxicity of several natural compounds including: okadaic acid, microcystin-LR, nodularin, calyculin A and tautomycin. To characterize further the actions of these toxins, this study compares the inhibitory effects of okadaic acid, chemical derivatives of okadaic acid, microcystin-LR, microcystin-LA, nodularin, calyculin A and tautomycin on the activity of serine/threonine protein phosphatases types 1 (PP1), 2A (PP2A) and a recently identified protein phosphatase purified from bovine brain (PP3). This study shows that, like PP1 and PP2A, the activity of PP3 is potently inhibited by okadaic acid, both microcystins, nodularin, calyculin A and tautomycin. Further characterization of the toxins employing the purified catalytic subunits of PP1, PP2A and PP3 under identical experimental conditions indicates that: (a) okadaic acid, microcystin-LR, and microcystin-LA inhibit PP2A and PP3 more potently than PP1 (order of potency PP2A > PP3 > PP1); (b) nodularin inhibits PP1 and PP3 at a similar concentration that is slightly higher than that which affects PP2A, and (c) both calyculin A and tautomycin show little selectivity among the phosphatases tested. This study also shows that the chemical modification of the (C1) carboxyl group of okadaic acid can have a profound influence on the inhibitory activity of this toxin. Esterification of okadaic acid, producing methyl okadaate, or reduction, producing okadaol, greatly decreases the inhibitory effects against all three enzymes tested. Further reduction, producing 1-nor-okadaone, or acetylation, producing okadaic acid tetraacetate, results in compounds with no inhibitory activity. In contrast, the substitution of alanine (-LA) for arginine (-LR) in microcystin has no apparent effect on the inhibitory activity against PP1, PP2A or PP3.

Reduced Connexin43 expression in high-grade human brain glioma cells

Connexin43 (cx43), a gap junction protein, is implicated in the suppression of tumor cell growth. Numerous cancer cells show a reduction or loss of cx43 expression compared to their normal counterparts. Our previous studies suggest that cx43 expression is decreased in a variety of human brain tumor cell lines. To further investigate the role of cx43 in the development of human gliomas, we performed the present study on human glioma grades I–IV.

Platelet-derived growth factor-induced disruption of gap junctional communication and phosphorylation of connexin43 involves protein kinase C and mitogen-activated protein kinase

Previously we showed a rapid and transient inhibition of gap junctional communication (GJC) by platelet‐derived growth factor (PDGF) in T51B rat liver epithelial cells expressing wild‐type platelet‐derived growth factor β receptors (PDGFrβ). This action of PDGF correlated with the hyperphosphorylation of the gap junction protein connexin43 (Cx43) and required PDGFrβ tyrosine kinase activity, suggesting the participation of protein kinases and phosphatases many of which are activated by PDGF treatment. In the present study, two such kinases, namely protein kinase C (PKC) and mitogen‐activated protein kinase (MAPK), are investigated for their possible involvement in PDGF‐induced closure of junctional channels and Cx43‐phosphorylation. Down‐regulation of PKC‐isoforms by 12‐O‐tetradecanoylphorbol‐13‐acetate or pretreatment with the PKC inhibitor calphostin C, completely blocked PDGF action on GJC and Cx43. Activation of MAPK correlated with PDGF‐induced Cx43 phosphorylation, and prevention of MAPK activation by PD98059 eliminated the PDGF effects. Interestingly, elimination of GJC recovery by cycloheximide was associated with a sustained activated‐MAPK level. Based on these results we postulate that the activation of PKC and MAPK are required in PDGF‐mediated Cx43 phosphorylation and junctional closure. J. Cell. Physiol. 176:332–341, 1998.

Effects of the tyrosine-kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells

We have examined the effects of the tyrosine kinase inhibitor genistein on hormone dependent cell proliferation and intracellular signalling in mouse 10T1/2 fibroblasts and rat liver T51B epithelial cells. Genistein inhibits both PDGF and EGF induced mitogenesis with an IC50 of 40 uM and 10 uM respectively. Genistein also inhibits inositol phosphate generation and calcium signalling in response to PDGF, and 1,2-diacylglycerol generation and calpactin II translocation in response to EGF. By contrast genistein does not inhibit inositol phosphate production, Ca2+ signalling or 1,2-diacylglycerol generation in response to ATP or angiotensin II. These data demonstrate that genistein selectively inhibits tyrosine kinase dependent processes without effecting similar responses obtained to hormones which are not dependent upon tyrosine kinase activation.

Rapid disruption of gap junctional communication and phosphorylation of connexin43 by platelet‐derived growth factor in T51B rat liver epithelial cells expressing platelet‐derived growth factor receptor

Gap junctional communication (GJC) between contacting cells has been postulated to be involved in the regulation of cell proliferation. This suggestion stems from numerous studies showing modulation of GJC by agents that influence cellular proliferation. Platelet‐derived growth factor (PDGF), a strong mitogen, inhibits GJC in many cell types. To understand the molecular nature of the signal transduction pathway responsible for the GJC blockade, T51B rat liver epithelial cells, which lack endogenous PDGF receptor (PDGFr), were infected with a retrovirus containing either wild‐type full‐length cDNA of human PDGFrβ (Kin+) or a mutant PDGFrβ lacking receptor tyrosine kinase activity (Kin−). PDGF caused a complete but transient interruption of cell communication in Kin+ cells within 15–20 min of addition. This interruption of GJC was not associated with a gross destabilization of gap junction plaques but with the phosphorylation of connexin43 (Cx43), the only known gap junction protein expressed in these cells. These effects were not exhibited in either control T51B cells or in Kin− cells, indicating a requirement of the receptor tyrosine kinase activity. Further examination revealed that the newly phosphorylated Cx43 then undergoes a rapid degradation utilizing the lysosomal pathway resulting in a decreased total Cx43 protein level. The re‐establishment of GJC following PDGF treatment was dependent on protein synthesis. This report describes a suitable cell system which is currently being utilized for the characterization of the PDGF signaling pathway responsible for the inhibition of GJC. J. Cell. Physiol. 174:66–77, 1998.

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet‐derived growth factor (PDGF)–induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild‐type PDGF receptor β (PDGFrβ). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen‐activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF‐ or TPA‐induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF‐ and TPA‐induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2‐mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N‐acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC. J. Cell. Physiol. 179:87–96, 1999.

Microcystin-LA from a blue-green alga belonging to the stigonematales

Abstract Microcystin-LA accounts for the protein phosphatase (types 1, 2A and 3) inhibitory activity of the terrestrial blue-green alga Hapalosiphon hibernicus BZ-3-1.

Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells

Gap junctional communication (GJC) has been implicated in the control of cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their normal counterparts. Lack of adequate information on the status of gap junctions during prostate neoplasia prompted us to examine this form of cancer, which comprises about 14% of male cancer deaths in America.

UV irradiation upregulates Egr‐1 expression at transcription level

UVC irradiation rapidly and strongly induces protein expression of the early growth response‐1 gene (Egr‐1) encoding a transcription factor which may have a protective function against UV damage. In this paper, we further investigate mechanisms responsible for such induction. We show that UVC irradiation also induced Egr‐1 mRNA expression, increased transcription rate by nuclear run‐on assay and stimulated Egr‐1 promoter activity by CAT assay. The Egr‐1 mRNA stability remained unchanged in UVC‐treated cells. On the other hand, UVC irradiation slightly extended Egr‐1 protein half‐life. The induction of Egr‐1 by UVC was observed in many different cell types. UVA and UVB also strongly induced Egr‐1 expression. These results indicate that UVC regulates Egr‐1 expression at transcription level. The induction pattern of Egr‐1 by UV suggests the importance of Egr‐1 in the UV response. J. Cell. Biochem. 73:227–236, 1999.

Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells.

The microbial toxin okadaic acid (OA) specifically inhibits PPP‐type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53‐mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor‐promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non‐neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53‐regulated gene products whose induction was blocked by siRNA‐mediated knockdown of p53. As observed with 10 nM OA, both phospho‐ser15‐p53 levels and p53 activity were increased by 10 µM fostriecin or SV40 small t‐antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA‐mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral‐mediated over‐expression of an OA‐resistant form of PP5 did not prevent increased phospho‐ser15‐p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA‐induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53‐related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA. J. Cell. Biochem.