Shedding of CD163, a novel regulatory mechanism for a member of the scavenger receptor cysteine-rich family

The glucocorticoid-inducible transmembrane protein CD163 is a member of the scavenger receptor cysteine-rich (SRCR) family which is expressed exclusively on human monocytes and macrophages. The expression of the protein is significantly downregulated in response to phorbol 12-myristate 13-acetate (PMA) by a yet unknown mechanism. We now demonstrate that PMA induces shedding of a soluble form of CD163 rather than internalization, revealing a novel regulatory mechanism for a member of the SRCR family. Bisindolylmaleimide I was shown to inhibit phorbol ester-induced shedding, thus implying an involvement of protein kinase C (PKC). Furthermore, cleavage could be prevented by protease inhibitors. Therefore, we suggest that PMA-induced activation of PKC leads to protease-mediated shedding of CD163. These results indicate a specific release mechanism of soluble CD163 by human monocytes which could play an important role in modulating inflammatory processes.     

Tumor necrosis factor-alpha-converting enzyme controls surface expression of c-Kit and survival of embryonic stem cell-derived mast cells

Transmembrane metalloproteinases of the disintegrin and metalloproteinase (ADAM) family control cell signaling interactions via hydrolysis of protein extracellular domains. Prior work has shown that the receptor tyrosine kinase, c-Kit (CD117), is essential for mast cell survival and that serum levels of c-Kit increase in proliferative mast cell disorders, suggesting the existence of c-Kit shedding pathways in mast cells. In the present work, we report that tumor necrosis factor alpha-converting enzyme (TACE; ADAM-17) mediates shedding of c-Kit. Stimulation of transfected cells with phorbol 12-myristate 13-acetate (PMA) induced metalloproteinase-mediated release of c-Kit ectodomain, which increased further upon TACE overexpression. By contrast, TACE-deficient fibroblasts did not demonstrate inducible release, thus identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface expression of c-Kit by the human mast cell-1 line decreased upon phorbol-induced shedding, which involved metalloproteinase activity susceptible to inhibition by tissue inhibitor of metalloproteinase (TIMP)-3. To further explore the role of TACE in shedding of c-Kit from mast cells, we compared the behavior of mast cells derived from murine embryonic stem cells. In these studies, PMA decreased surface c-Kit levels on mast cells expressing wild-type (+/+) TACE but not on those expressing an inactive mutant (DeltaZn/DeltaZn), confirming the role of TACE in PMA-induced c-Kit shedding. Compared with TACE(+/+) cells, TACE(DeltaZn/DeltaZn) mast cells also demonstrated decreased constitutive shedding and increased basal surface expression of c-Kit, with diminished apoptosis in response to c-Kit ligand deprivation. These data suggest that TACE controls mast cell survival by regulating shedding and surface expression of c-Kit.     

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17)

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.     

Regulated cell surface pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process

Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.     

PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.     

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 μM), 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 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM 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 μM 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. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade.

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.     

Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G

There are two known phosphorylation-mediated inactivation mechanisms for TRPC3 channels. Protein kinase G (PKG) inactivates TRPC3 by direct phosphorylation on Thr-11 and Ser-263 of the TRPC3 proteins, and protein kinase C (PKC) inactivates TRPC3 by phosphorylation on Ser-712. In the present study, we explored the relationship between these two inactivation mechanisms of TRPC3. HEK cells were first stably transfected with a PKG-expressing construct and then transiently transfected with a TRPC3-expressing construct. Addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG), elicited a TRPC3-mediated [Ca2+]i rise in these cells. This OAG-induced rise in [Ca2+]i could be inhibited by phorbol 12-myristate 13-acetate (PMA), an agonist for PKC, in a dose-dependent manner. Importantly, point mutations at two PKG phosphorylation sites (T11A-S263Q) of TRPC3 markedly reduced the PMA inhibition. Furthermore, inhibition of PKG activity by KT5823 (1 microM) or H8 (10 microM) greatly reduced the PMA inhibition of TRPC3. These data strongly suggest that the inhibitory action of PKC on TRPC3 is partly mediated through PKG in these PKG-overexpressing cells. The importance of this scheme was also tested in vascular endothelial cells, in which PKG plays a pivotal functional role. In these cells, OAG-induced [Ca2+]i rise was inhibited by PMA, which activates PKC, and by 8-BrcGMP and S-nitroso-N-acetylpenicillamine (SNAP), both of which activate PKG. Importantly, the PMA inhibition on OAG-induced [Ca2+]i rise was significantly reduced by PKG inhibitor KT5823 (1 microM) or DT-3 (500 nM), suggesting an important role of PKG in the PMA-induced inhibition of TRPC channels in native endothelial cells.     

Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ϵ, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.     

Transforming growth factor-alpha and beta-amyloid precursor protein share a secretory mechanism

Cleavage and release of membrane protein ectodomains, a regulated process that affects many cell surface proteins, remains largely uncharacterized. To investigate whether cell surface proteins are cleaved through a shared mechanism or through multiple independent mechanisms, we mutagenized Chinese hamster ovary (CHO) cells and selected clones that were unable to cleave membrane-anchored transforming growth factor alpha (TGF-alpha). The defect in TGF-alpha cleavage in these clones is most apparent upon cell treatment with the protein kinase C (PKC) activator PMA, which stimulates TGF-alpha cleavage in wild-type cells. The mutant clones do not have defects in TFG-alpha expression, transport to the cell surface or turnover. Concomitant with the loss of TGF-alpha cleavage, these clones have lost the ability to cleave many structurally unrelated membrane proteins in response to PMA. These proteins include beta-amyloid precursor protein (beta-APP), whose cleavage into a secreted form avoids conversion into the amyloidogenic peptide A beta, and a group of cell surface proteins whose release into the medium is stimulated by PMA in wild type CHO cells but not in mutants. The mutations prevent cleavage by PKC- dependent as well as PKC-independent mechanisms, and thus affect an essential component that functions downstream of these various signaling mechanisms. We propose that regulated cleavage and secretion of membrane protein ectodomains is mediated by a common system whose components respond to multiple activators and act on susceptible proteins of diverse structure and function.     

Tachykinin-Stimulated Inositol Phospholipid Hydrolysis and Taurine Release from Human Astrocytoma Cells

The activation of NK1 receptors on U373 MG human astrocytoma cells by substance P (SP) and related tachykinins was accompanied by an increase in taurine release and an accumulation of inositol phosphates. Both of these effects could be inhibited by spantide, a SP receptor antagonist. The relative potency of tachykinins in stimulating 3H-inositol phosphate accumulation correlated very well with their effects in stimulating the release of [3H]-taurine and inhibition 125I-Bolton-Hunter reagent-conjugated SP binding. The effect on [3H]taurine release was mimicked by a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). The inactive phorbol ester analogue 4-alpha-phorbol 12,13-didecanoate, however, was without effect. Both SP- and PMA-induced releases of [3H]-taurine were markedly inhibited by staurosporine, a potent PKC inhibitor. Pretreatment of U373 MG cells with 10 microM PMA for 19 h to down-regulate PKC activity also markedly inhibited both SP- and PMA-induced releases of [3H]-taurine. Treatment of cells with 100 nM SP induced a time-dependent translocation of PKC from the cytosolic fraction to the membrane fraction. These findings are consistent with the hypothesis that an activation of NK1 receptors on U373 MG cells results in the release of inositol phosphates and activation of PKC, which in turn may regulate the release of taurine.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells.

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.     

Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1)

Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.     

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.     

Differential effects of forskolin and phobol 12-myristate-13-acetate on the c-fos and c-jun mRNA expression in rat C6 glioma cells

The effects of forskolin (FSK) and phobol 12-myristate-13-acetate (PMA) on c-fos and c-jun mRNA expressions in rat C6 glioma cells were studied. Both FSK and PMA increased the c-fos mRNA level. The C-jun mRNA level was decreased by FSK, whereas it was increased by PMA. The elevated c-fos mRNA level, induced by FSK or PMA, was significantly inhibited by dexamethasone (DEX). In contrast, DEX did not affect the FSK- and PMA-induced response of the c-jun mRNA level. Cycloheximide (CHX) caused a superinduction of the FSK- or PMA-induced c-fos mRNA level. Furthermore, CHX also potentiated the PMA-induced c-jun mRNA level. However, CHX did not affect the FSK-induced down-regulation of the c-jun mRNA level. When C6 glioma cells were incubated with PMA and FSK, the PMA-induced c-jun mRNA level was inhibited by FSK, whereas FSK did not affect the PMA-induced c-fos mRNA level. Our results suggest that the activations of PKA and PKC pathways have different roles in the regulation of the c-jun mRNA expression in rat C6 glioma cells. PKA activation can inhibit induction of the c-jun mRNA expression by PMA. In addition, DEX appears to have a selective inhibitory action against c-fos, but not c-jun, -mRNA expression that is regulated by PKA and PKC. On-going protein synthesis inhibition is required for the superinduction of the c-fos expression that is induced by PMA, or FSK and the PMA-induced c-jun mRNA level.