PKC Signaling Pathways Research Articles

Calcitonin induces IL-6 production via both PKA and PKC pathways in the pituitary folliculo-stellate cell line.

It has been demonstrated that calcitonin-binding sites are present in a variety of tissue types, including in the pituitary gland. Interleukin-6 (IL-6) is also produced in the pituitary and it regulates the secretion of various hormones. In this study, we examined the expression of the calcitonin receptor and the mechanism of IL-6 production induced by calcitonin in the pituitary folliculo-stellate cell line (TtT/GF). The mRNA of calcitonin receptor subtype C1a, but not that of C1b, was detected by RT-PCR in TtT/GF cells and in the normal mouse pituitary. Calcitonin increased cAMP accumulation and IL-6 production in a concentration-dependent manner in TtT/GF cells. As calcitonin activates the PKA and PKC pathways, we investigated the contributions of PKA and PKC to IL-6 production. IL-6 production was only slightly increased by either 8-bromo-cAMP (1 mM) or phorbol 12-myristate 13-acetate (100 nM) alone. However, IL-6 was synergistically induced in the presence of both 8-bromo-cAMP (1 mM) and phorbol 12myristate 13-acetate (100 nM). Furthermore, calcitonin-induced IL-6 production was completely suppressed by H-89 (PKA inhibitor) or GF109203X (PKC inhibitor), indicating that the activation of both PKA and PKC is necessary for calcitonin-induced IL-6 production. On the other hand, pertussis toxin (G(i)/G(o) signaling inhibitor) treatment achieved an approximately 9-fold increase in calcitonin-induced IL-6 production. These results show that calcitonin-stimulated IL-6 production is mediated via both PKA- and PKC-signaling pathways, whereas calcitonin also suppresses IL-6 production by activating G(i)/G(o) proteins in folliculo-stellate cells.

Protein kinase C signalling pathway is involved in the regulation of vascular endothelial growth factor expression in human bladder transitional carcinoma cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the growth and metastasis of various cancers and plays a prominent role in vesical angiogenesis regulation. In this study, we investigated the effect of the phorbol 12-myristate 13-acetate (PMA) on the expression of VEGF in human bladder transitional carcinoma cells (RT4). RT4 cells expressed three VEGF isoforms (VEGF 189, VEGF 165, VEGF 121). PMA increased VEGF mRNA expression time-dependently with a peak at 4 h. PMA increased the half-life of VEGF mRNA. The amount of VEGF protein in conditioned media was increased by PMA in a dose-dependent manner with a maximal effect at 10 −7 M. Staurosporine and calphostin C (PKC inhibitors) decreased PMA-induced VEGF mRNA expression as opposed to protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Thus, in RT4 cells, VEGF expression is up-regulated by PMA via the PKC signalling pathway and according to a posttranscriptional mechanism.

Involvement of p-15INK4b and p-16INK4a Gene Expression in Saikosaponin a and TPA-Induced Growth Inhibition of HepG2 Cells

Saikosaponin a, a purified ingredients of Chinese herb with known antitumor activity can inhibit cell growth and DNA synthesis of hepatoma cell line HepG2. Both mRNA and protein of the CDK inhibitor p-16INK4a and p-15INK4b in HepG2 were greatly induced by saikosaponin a while that of p-21CIP, p-27KIP and other cell cycle related genes were not. In addition, reduced phosphorylation of RB protein is observed in saikosaponin a-treated HepG2. Staurosporin, one of the PKC inhibitors, significantly prevented the saikosaponin a induced growth inhibition suggesting PKC pathway be involved. On the other hand, the phorbol ester tumor promoter TPA (12-O-Tetredecanolyphorbol 13-acetate) also inhibited HepG2 growth and specifically induced p-16INK4a and p-15INK4b mRNA expression. The results suggest that both saikosaponin a and TPA-induced HepG2 growth inhibition are associated with p-15INK4a and p-16INK4b gene expression and might be mediated by PKC signaling pathway.

Possible Molecular Targets of Halogenated Aromatic Hydrocarbons in Neuronal Cells

Halogenated aromatic hydrocarbon including polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental toxicants. Although health effects associated with exposure to these chemicals, including motor dysfunction and impairment in memory and learning, have been identified, their molecular site of action is unknown. Previous study from this laboratory demonstrated that, while ortho PCBs perturbed intracellular signaling mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production and translocation of PKC, non-ortho PCBs did not. Since PKC signaling pathway is implicated in the modulation of motor behavior, as well as learning and memory, and the roles of PKC are isoform-specific, we have now studied the effects of two structurally distinct PCBs on isoforms of PKC in cerebellar granule cell culture model. Cells were exposed to 2,2′-dichlorobiphenyl (ortho PCB; 2,2′-DCB) or 4,4′-dichlorobiphenyl (non-ortho PCB; 4,4′-DCB) for 15 min, respectively, and subsequently fractionated and immunoblotted against the selected PKC monoclonal antibodies (α, γ, δ, ϵ, λ, ι). While 2,2′-DCB induced a translocation of PKC-α [cytosol (% control): 54 ± 12 at 25 μM and 66 ± 10 at 50 μM; membrane (% control): 186 ± 37 at 25 μM and 200 ± 48 at 50 μM] and −ϵ [cytosol (% control): 92 ± 12 at 25 μM and 97 ± 15 at 50 μM; membrane (% control): 143 ± 23 at 25 μM and 192 ± 24 at 50 μM] from cytosol to membrane fraction in a concentration-dependent manner, 4,4′-DCB had no effects. 2,2′-DCB induced translocation of PKC-α was blocked by pretreatment with sphingosine, suggesting a possible role of sphingolipid pathway. Although reports on implication of PKC-γ with learning and memory are relatively extensive, the expression of this particular isoform in the primary cerebellar granule cells was below the detectable level. PKC-δ, -λ and -ι were present in these cells, but were not altered by PCB exposure. These results suggest that the effects of 2,2′-DCB on PKC is isoform-dependent and PKC-α as well as PKC-ϵ may be target molecules for ortho-PCBs in neuronal cells.

Angiogenesis in gastric microvascular endothelial cells is dependent on the MAPK, PI-3K and PKC signaling pathways

Angiogenesis in gastric microvascular endothelial cells is dependent on the MAPK, PI-3K and PKC signaling pathways

Inducible expression of manganese superoxide dismutase by phorbol 12-myristate 13-acetate is mediated by Sp1 in endothelial cells.

The expression of manganese superoxide dismutase (Mn-SOD), an important component of the cellular defense system against oxidative stress, is induced in response to a variety of stimuli, including cytokines and phorbol esters, in endothelial cells. To define the molecular mechanisms regulating the expression of Mn-SOD, we have characterized the promoter of the human Mn-SOD gene. In calf pulmonary artery endothelial cells, phorbol 12-myristate 13-acetate (PMA) gradually increased Mn-SOD mRNA levels, with a peak at 6 to 12 hours after stimulation. The increase in Mn-SOD mRNA was significantly inhibited by a protein kinase C (PKC) inhibitor (calphostin C) but not by a mitogen-activated protein kinase kinase-1 inhibitor (PD98059) or a p38 mitogen-activated protein kinase inhibitor (SB203580). By reporter gene transfection experiments of a series of promoter deletions and site-directed mutation constructs, we found 2 consensus Sp1 binding sequences located at -97 and at -77 to play an important role in PMA-induced Mn-SOD transcription. Electrophoretic gel mobility shift assays have indicated that this sequence serves as an Sp1 binding site. Northern and Western blot analysis has revealed that PMA-induced promoter activity of Mn-SOD correlates with an increased expression of Sp1. Nuclear proteins from PMA-treated calf pulmonary artery endothelial cells displayed an increased DNA binding to the Sp1 site. Furthermore, the Mn-SOD promoter was activated either by overexpression of Sp1 or the constitutively activated form of PKCbeta in an Sp1 site-dependent manner. These results suggest that PMA stimulates transcription of the Mn-SOD gene through an increase in Sp1 expression and thus implicate Sp1 as an effector mediating the PKC-signaling pathway elicited by extracellular signals.

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells.

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a beta1/beta2-adrenergic receptor (AR) agonist) and iodoclonidine (an alpha2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (alpha1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (beta-AR blocker), atenolol (beta1-AR blocker), yohimbine (alpha2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (beta2-AR blocker) and prazosin (alpha1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5'-flanking region of the ANG gene and subsequently stimulates the gene expression.

Inhibition of Tissue Factor Synthesis by Disruption of ERK Kinases and PKC Signaling Pathways in Human Vascular SMCs

Tissue Factor (TF), the receptor for plasma VII/VIIa and the initiator of blood coagulation, is inducible in vascular smooth muscle cells (SMCs) by growth factors and bacterial lysopolysaccharides (LPS) and is expressed in vivo after vascular injury. As TF expression is a determinant of the thrombogenicity of vascular lesions, we investigated the signal pathways involved in this process. Human vascular SMCs were obtained from normal arteries and made quiescent by serum deprivation. Baseline TF antigen and activity were up-regulated by various agonists: fetal calf serum (FCS), LPS, and platelet derived growth factor (PDGF) being the most effective but with different kinetics. TF expression induced by LPS was transient with a maximum 6 h after stimulation and returned to baseline levels after 24 h whereas TF expression induced by serum or PDGF was sustained for at least 24 h. Rapid and transient activation of Extracellular signal-Regulated Kinase (ERK) was observed after stimulation by PDGF and FCS, but not by LPS. The role of ERK, Ras and protein kinase C activities were investigated using specific inhibitors, PD 98059, manumycin A and calphostin C respectively. For TF induction by LPS, PKC activity was required and the ERK/Ras pathway was not involved. In contrast, the effect of PDGF was strictly ERK and Ras dependent, but partially prevented by PKC inhibitors. TF induction by FCS was ERK dependent but partially Ras and PKC dependent. In conclusion, TF expression appears to be a non-specific response of SMCs to numerous stimuli through multiple signal pathways which differ according to the inducing agent.

Mechanism of inhibition of P-glycoprotein-mediated drug transport by protein kinase C blockers

P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport by mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.

Concerted regulation of wild-type p53 nuclear accumulation and activation by S100B and calcium-dependent protein kinase C.

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G(1) checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272-4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G(1) phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G(1) arrest at the nonpermissive temperature (37.5 degrees C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32 degrees C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G(1) phase and activation of a p53-dependent G(1) checkpoint control.

Myocardial adaptation to ischaemia – the preconditioning phenomenon

The phenomenon of ischaemic preconditioning, highlights a new and endogenous route to myocardial protection, which we believe could be exploited in our search for new therapeutic ways to protect the infarcting myocardium. Ischaemic preconditioning has been shown to be associated with both an early, or acute phase of protection lasting approximately 1–2 hours, as well as a delayed phase or “second window of protection” seen at least 24 hours following the initial sublethal ischaemic insult, and lasting up to 72 hours. We believe that both responses are triggered by similar receptor mediated events in addition to using the similar signalling pathways involving kinase cascades. However it is thought that the ultimate target or end-effector throught which the protection is manifest may be different for the early vs. late effects. Some evidence exists that the end-effector involved in early preconditioning may be via the ATP-sensitive potassium channel (K ATP). With respect to the second window of protection, the cellular mechanisms underlying this are not fully understood at present, however we believe that they may be dependent upon a similar signalling transduction pathway with upregulation of cytoprotective proteins such as the heat stress proteins, and/or anti-oxidant proteins. Evidence demonstrating that preconditioning can occur in the human myocardium is also accumulating. In this respect cultured human ventricular myocytes as well as human atrial muscle have been shown to be preconditioned with brief episodes of simulated ischemia. These human preparations also respond to the known triggers and possible end-effectors of preconditioning, (e.g. adenosine receptor stimulation and K ATP channel opening) as well as being able to elicit their responses through the PKC signalling pathway. Further support for this phenomenon, in man, comes from PTCA studies demonstrating that this invasive procedure can put patients into a “preconditioned state”; this effect being associated with reduced ischaemic symptoms as well as the involvement of the adenosine receptor and K ATP channel. Of further interest is the observation that patients with a previous history of angina, prior to a MI, sustain smaller infarcts and have an improved survival. However the most direct evidence that preconditioning occurs in man comes from studies in partients undergoing coronary artery bypass surgery. The above evidence that preconditioning can occur in man makes it now possible to begin to design clinical studies investigating cardioprotective properties of drugs that can specifically mimic this phenomenon.

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation.

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.

Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae.

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.

Carbon source‐dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECε) coding for the expression of murine protein kinase C epsilon (PKCε) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECε expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCε antibody. Extracts from these pYECε-transfected cells could phosphorylate a PKCε substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCε was fused in frame with GST (GST-Rε), further confirming the successful expression of murine PKCε. Induction of PKCε expression by galactose in cells transformed with pYECε increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCε expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCα or PKCβ-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCε expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCε expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCε-expressing yeast cells (1–2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCε expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCε expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCε-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells. J Cell Physiol 178:216–226, 1999. © 1999 Wiley-Liss, Inc.

Evaluation of hormonal response of bovine granulosa cells following activation of PKA or PKC signaling pathways

Evaluation of hormonal response of bovine granulosa cells following activation of PKA or PKC signaling pathways

Basic Fibroblast Growth Factor Release by Human Coronary Artery Endothelial Cells Is Enhanced by Matrix Proteins, 17β-Estradiol, and a PKC Signaling Pathway

Endothelial cell function is regulated by interactions among cells, the extracellular matrix (ECM), and soluble mediators. We investigated this interaction by examining the effect of 17β-estradiol (E2) on release of basic fibroblast growth factor (FGF-2) by human coronary artery endothelial cells (HCAEC) cultured on ECM proteins. After estrogen-depleted HCAEC were treated with E2 for 2 h, the conditioned media and cell layers were evaluated by immunoblot or ELISA for FGF-2. Release of FGF-2 into conditioned media was enhanced 10-fold compared to that on plastic and a further 2.4-fold by E2. As FGF-2 release from cells into the media increases, there is a corresponding decrease in the cellular content of FGF-2. By ELISA, FGF-2 release increased 406, 179, and 262%, on type IV collagen, laminin, or fibronectin, respectively. HCAEC cultured on type I collagen did not show E2-enhanced FGF-2 release by ELISA or immunoblot analysis. No changes were noted in HCAEC release of lactate dehydrogenase, tested as a control protein for cellular integrity. The estrogen receptor antagonist ICI182,780 blocked E2-induced, but not basal, FGF-2 release. Increased FGF-2 release occurred via a cycloheximide-insensitive pathway. Neither brefeldin-A nor genistein inhibited E2 enhancement of FGF-2 release by HCAEC cultured on fibronectin. However, the protein kinase C inhibitor calphostin C inhibited the E2-augmented FGF-2 release. These data show that E2 enhances FGF-2 release by HCAEC cultured on basement membrane proteins in the absence of wounding. This action requires the estrogen receptor and PKC activity, but does not require new protein synthesis, endoplasmic reticulum-to-Golgi-mediated secretion, or protein tyrosine phosphorylation. E2-enhanced FGF-2 release could contribute to the cardioprotective effects of estrogen.

Transcriptional regulation of fibroblast growth factor-2 expression in human astrocytes: implications for cell plasticity.

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Acetylcholine-Induced Biphasic Effect on the Maximum Upstroke Velocity of Action Potential in Mouse Right Atria: Interaction With β-Adrenergic Signaling Cascade

Several lines of evidence suggest the molecular and functional entity of muscarinic M1 receptors in mammalian heart. We have reported that acetylcholine (ACh) reduces the maximum upstroke velocity of action potential (Vmax) through activation of muscarinic M1 receptors, which is followed by a muscarinic M2 receptor-mediated increase. The present study sought to determine whether activation of β-adrenergic receptors modulates the muscarinic M1 and M2 receptor-mediated effects on Vmax in isolated mouse right atria. Intracellular recordings of spontaneous action potential were done using the conventional glass microelectrode technique. Isoproterenol (3 nM) completely antagonized ACh (5 μM)-induced reduction in Vmax. The antagonism was accompanied by a subsequent increase in Vmax. Propranolol (0.3 μM) abolished the effects of isoproterenol on ACh-induced changes in Vmax. Isoproterenol antagonized McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride) (300 μM, a muscarinic M1 receptor agonist)-induced reduction in Vmax. Oxotremorine (0.03 μM), a muscarinic M2 receptor agonist, did not affect Vmax by itself, but significantly increased it in the presence of 3 nM isoproterenol. The effects of isoproterenol were mimicked by cholera toxin (100 nM, 1 hr), a Gs-protein activator, and forskolin (10 nM), a direct activator of adenylyl cyclase. H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide, 1 μ/M), a selective protein kinase (PK)-A inhibitor, abolished the antagonism by isoproterenol of ACh-induced reduction in Vmax. The present results suggest that activation of the β-adrenergic-Gs-adenylyl cyclase system antagonizes ACh-induced reduction (muscarinic M1-mediated) and potentiates the subsequent increase (muscarinic M2 receptor-mediated) in Vmax. The β-adrenergic antagonism of ACh-induced reduction in Vmax may involve cross-talk between PK-A and PK-C signaling pathways.

Protein phosphatase-1 and insulin action.

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF-alpha (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1G subunit site-specific mutations will help clarify the exact role and regulation of PP-1G site-specific phosphorylations on PP-1 catalytic function.

Urinary trypsin inhibitor, a Kunitz-type protease inhibitor, modulates tumor necrosis factor-stimulated activation and translocation of protein kinase C in U937 cells.

Urinary trypsin inhibitor (UTI) is a Kunitz-type protease inhibitor. We have reported that UTI inhibited TNF-induced urokinase (uPA) production via a protein kinase C (PKC)-dependent mechanism. It is likely that UTI suppresses tumor cell invasion and metastasis by a mechanism, possibly by inhibiting uPA production. In the present study, we attempted to determine how UTI is associated with PKC, and how UTI is involved in uPA-dependent tumor cell invasion and metastasis. The increments of membrane-associated PKC activity by TNF were subsequently accompanied by a rapid loss of cytosol-associated PKC activity in U937 leukemia cells. Semi-quantitative immunoblotting of membrane and cytosol fractions showed that the translocation of PKC-alpha, -beta, and -epsilon were blocked by the addition of UTI in cells stimulated with TNF but not in cells stimulated with PMA, demonstrating that PKC itself is not sensitive to UTI. This effect was dependent on the carboxyl-terminus of UTI. In addition, UTI neither inhibited TNF binding to cellular receptors nor inactivate PKC and uPA activities directly. Taken together, the experiments suggest that the carboxyl-terminus of UTI may inhibit the PKC-signalling pathways upstream of diacylglycerol by a mechanism, possibly by interrupting the coupling of receptor and effector systems. UTI was shown to have an interesting new function besides being a protease inhibitor. This is the first report that UTI has a selective inhibition of TNF-activated PKC. We conclude that UTI suppresses tumor cell invasion and metastasis by a mechanism that UTI inhibits TNF-stimulated uPA production via a PKC-dependent mechanism.