Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.     

Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.     

Mode of action of prostaglandin F2 alpha in human luteinized granulosa cells: role of protein kinase C

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.     

Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.     

Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively. PMA at 1 microM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCbeta) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane. Western blot analysis revealed the presence of eight PKC isoforms (alpha, betaI, betaII, delta, epsilon, mu, lambda and xi) in RAW 264.7 cells and PMA was shown to induce the translocation of the alpha, betaI, betaII, delta, epsilon and mu isoforms from the cytosol to the cell membrane within 2 min. Pretreatment of cells with PMA for 2-24 h resulted in a time-dependent down-regulation of PKCalpha, betaI, betaII, and delta expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC. These results indicate that PKCbeta has a crucial role in the mediation of cPLA2 activation by the phorbol ester PMA, whereas PMA utilizes PKC epsilon and/or mu to up-regulate AC activity.     

Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation

Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the beta1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin-containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin alphaLbeta2. Although C3 treatment of PB T cells did not prevent adhesion to the beta1 integrin substrate Fn, it did inhibit beta1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited beta1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.     

P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.     

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion.     

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.     

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.     

Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule

The ability of the CD44 adhesion molecule to interact with its ligand hyaluronic acid (HA) is tightly regulated. CD44-positive mouse LB lymphoma cells are unable to bind HA unless activated by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PMA causes a dose-dependent increase in both CD44 expression level and HA-binding capacity, with the binding of HA observed only above a threshold amount of CD44 molecules. This induction of HA-binding as well as the increase in CD44 expression are prevented by cycloheximide, suggesting a requirement for new additional CD44 molecules on the cell surface and/or cooperating proteins. In the present study, we have investigated which of the signal transduction pathways activated by PMA leads to the increased CD44 expression with subsequent acquisition of HA-binding capacity. By comparing the influence of each inhibitory agent on PMA-activated LB lymphoma cells versus that on a constitutive HA-binder cell line derived from LB cells (designated HA9 cells), we could distinguish between an effect on the PMA-activation phase and a one on the HA-binding phase. Our data show that the PMA-induced HA-binding could not be blocked by agents inhibiting protein kinase C (PKC) (staurosporine, sphingosine, polymyxin B, quercetin) or genestein, an inhibitor of tyrosine protein kinases. However, this PMA response was strongly inhibited by calmodulin antagonists (chlorpromazine, trifluoperazine, W-7) and the calcium blocker verapamil. The calmodulin antagonists inhibited the PMA-induced increase in CD44 expression on LB cells, but had no influence on the ability of the constitutive HA-binder HA9 cell line to interact with HA, indicating an effect on the PMA induction phase rather than on the binding itself. Verapamil also blocked the PMA-induced increase in CD44 expression on LB cells, but in addition it slightly reduced the ability of the HA9 cells to bind HA without affecting their CD44 expression level. In conclusion, our data suggest that CD44 activation by PMA is calcium and calmodulin dependent, rather than mediated by protein kinase C.     

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.     

Porphyromonas gingivalis fimbriae use beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as a cellular receptor, and the CD18 beta chain plays a functional role in fimbrial signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the beta chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) genes in the cells. Using a binding assay with 125I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of beta2 integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of beta2 integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.     

C2 region-derived peptides of beta-protein kinase C regulate cardiac Ca2+ channels

We have previously shown that alpha1-adrenergic activation inhibited beta-adrenergic-stimulated L-type Ca2+ current (I(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4beta-phorbol 12-myristate 13-acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 micromol/L) consistently inhibited basal I(Ba) by 40.5+/-7.4% and isoproterenol (ISO, 0.1 micromol/L)-stimulated I(Ba) by 48.9+/-7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue alpha-phorbol 12,13-didecanoate (0.1 micromol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (betaC2-2 and betaC2-4) specifically inhibit the translocation and function of C2-containing isozymes (alpha-PKC, betaI-PKC, and betaII-PKC), but not the C2-less isozymes (delta-PKC and epsilon-PKC). We first used the pseudosubstrate peptide (0.1 micromol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ba) was reduced to 16.8+/-7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2-derived peptides or control peptides. When the pipette contained 0.1 micromol/L of betaC2-2 or betaC2-4, PMA-induced inhibition of basal I(Ba) was 26.1+/-4.5% and 23.6+/-2.2%, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9+/-6.6% and 29.3+/-7.8% in the presence of betaC2-2 and betaC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled betaC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both betaC2-2 and betaC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.     

Rapid adhesion and spread of non-adherent colon cancer Colo201 cells induced by the protein kinase inhibitors, K252a and KT5720 and suppression of the adhesion by the immunosuppressants FK506 and cyclosporin A

We examined alterations in cell morphology and expression of adhesion molecules in response to a general protein kinase inhibitor K252a treatment of non-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell adhesion and spreading with concomitant formation of actin stress fibers. A protein kinase A inhibitor KT5720 also induced cell adhesion, but the rate of spread was slower than that seen with K252a. These adhesions were mediated by integrin molecules since cell adhesion required Mg2+, Mn2+ or Ca2+, and was inhibited by monoclonal antibodies for integrins alpha2 and beta1. Indirect immunofluorescence microscopic observations revealed that integrin alpha2 and beta1 molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin increased during K252a- or KT5720-induced cell adhesion. Immunosuppressants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion and abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, also inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.     

Stimulation of human B lymphocytes by phorbol esters reported to be selective in the protein kinase C isoforms they activate

It is reported here that B cells can be stimulated by two phorbol esters which, in cell free substrate phosphorylation assays, are selective in the PKC isoforms they activate: thymeleatoxin (Thy) stimulates all of the classical (c) or Group A PKCs (alpha, beta 1, beta 2 and gamma) but not PKC delta and epsilon which belong to the novel (n) or Group B PKCs, while 12-deoxyphorbol-13-O-phenylacetate-20-acetate (dPPA) is a specific activator of PKC beta 1. By itself, phorbol 12-myristate, 13-acetate (PMA)--which activates all cPKC and nPKC--was, on a molar basis, some 40-times more potent than either Thy or dPPA which were themselves equipotent at promoting DNA synthesis in resting B cells: the peak response achieved with Thy and dPPA was higher (1.4 x) than that obtained with PMA. In the presence of calcium ionophore, PMA, Thy and dPPA all stimulated a higher (and equivalent) peak response which was achieved at a lower phorbol ester concentration in each case: however, whereas Thy now approached PMA in potency, dPPA remained some 40-times less potent.