Intracellular Signaling Factors Research Articles

Fibronectin bound to the surface of Staphylococcus aureus induces association of very late antigen 5 and intracellular signaling factors with macrophage cytoskeleton.

Staphylococcus aureus Cowan I and a clinically isolated coagulase-negative Staphylococcus strain, S. saprophyticus 10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against S. aureus (by ca. 150%) but not against S. saprophyticus. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen 5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting fibronectin-bound Cowan I, alpha(5) and beta(1) chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated with the cytoskeleton. On the contrary, beta(3) integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated S. aureus Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components did not respond in a fibronectin-dependent manner when macrophages phagocytosed S. saprophyticus. This means that only fibronectin-treated S. aureus Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases. It is thought that the phagocytic activity of macrophages against fibronectin-treated S. aureus was increased by signaling via the activation of very late antigen 5.

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein.

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.

Gene expression for a novel protein RGPR-p117 in various species: the stimulation by intracellular signaling factors.

The presence and expression for the gene encoding a novel regucalcin gene promoter region-related protein (RGPR-p117) in various species was investigated by using Southern "zoo blot" and Northern hybridization analyses. A "zoo blot" analysis demonstrated that RGPR-p117 gene was widely conserved in various species including human, rat, mouse, dog, cow, pig, rabbit, chicken, fish, C. elegans and yeast. The gene was not found in Xenopus. Northern blot analysis showed that RGPR-p117 mRNA was expressed in the liver of human, rat, mouse, and rabbit as a single mRNA of approximately 4.5 kb, respectively. However, homologous mRNA was not found in the liver of Xenopus. The expression of RGPR-p117 mRNA in liver was clearly enhanced 5 h after a single intraperitoneal administration of CaCl(2) (5 mg Ca(2+)/100 g body weight) to rats. The RGPR-p117 mRNA is also expressed in the cloned H4-II-E rat hepatoma cells, although this expression was weak as compared with that of liver tissues. Moreover, the RGPR-p117 mRNA expression in H4-II-E cells was stimulated in the presence of dibutyryl cAMP, PMA, insulin, 17beta-estradiol, or serum in culture medium. The present study demonstrates that the RGPR-p117 gene is conserved in various species, and that its expression is stimulated by intracellular signaling factors.

Role of regucalcin as an activator of sarcoplasmic reticulum Ca2+-ATPase activity in rat heart muscle.

The expression of regucalcin, a regulatory protein of Ca(2+) signaling, and its effect on Ca(2+) pump activity in the microsomes (sarcoplasmic reticulum) of rat heart muscle was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in heart muscle using rat regucalcin-specific primers. Results with Western blot analysis showed that regucalcin protein was present in the cytoplasm, although it was not detected in the microsomes. Microsomal Ca(2+)-ATPase activity was significantly increased in the presence of regucalcin (10(-10)-10(-8) M) in the enzyme reaction mixture. This increase was not seen in the presence of thapsigargin (TP) (10(-5) M), a specific inhibitor of the microsomal Ca(2+) pump enzyme. Regucalcin (10(-10)-10(-8) M) significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. The effect of regucalcin (10(-8) M) in increasing microsomal Ca(2+)-ATPase activity was completely prevented in the presence of digitonin (10(-3) or 10(-2)%), which has a solubilizing effect on membranous lipid, or N-ethylmaleimide (NEM), a modifying reagent of sulfhydryl (SH) groups. Dithiothreitol (DTT; 5 mM), a protecting reagent of SH groups, increased markedly Ca(2+)-ATPase activity. In the presence of DTT (5 mM), regucalcin could not significantly enhance the enzyme activity. Also, the effect of regucalcin in increasing Ca(2+)-ATPase activity was completely inhibited by the addition of vanadate (1 mM), an inhibitor of phosphorylation of enzyme. In addition, the effect of regucalcin on Ca(2+)-ATPase activity was not significantly modulated in the presence of dibutyryl cyclic AMP (10(-4) M), inositol 1,4,5-trisphosphate (10(-3) M), or calmodulin (5 microg/ml) which is an intracellular signaling factor. The present study demonstrates that regucalcin can activate Ca(2+) pump activity in rat heart microsomes, and that the protein may act the SH groups of Ca(2+)-ATPase by binding to microsomal membranes.

Differential gene expression in the rat soleus muscle during early work overload-induced hypertrophy.

Delineating the molecular mechanisms that are responsive to work overload is crucial to understanding the adaptive processes controlling skeletal muscle mass. We have examined the molecular events associated with increased workload by using microarray analysis to begin to define the mechanotransduction responsive transcription programs in skeletal muscle. Microarray analysis identified 112 mRNAs that were expressed differentially in the soleus muscle of sham-operated vs. gastrocnemius-ablated rats. These genes can be classified into cell proliferation, autocrine/paracrine, extracellular matrix, immune response, intracellular signaling, metabolism, neural, protein synthesis/degradation, structural, and transcription. These findings dramatically increase the number of known, differentially expressed mRNA during early skeletal muscle hypertrophy. In toto, our findings indicate that work overload induced skeletal muscle hypertrophy alters autocrine/paracrine signaling, intracellular signaling, and transcription factor expression, which likely results in a dramatic change in cellular metabolism, cell proliferation, and muscle structure. These data enhance our understanding of the complex molecular mechanisms controlling skeletal muscle mass in response to increased physical activity.

MAP Kinase Converts MyoD into an Instructive Muscle Differentiation Factor in Xenopus

In amphibian development, muscle is specified in the dorsal lateral marginal zone (DLMZ) of the gastrula embryo. Two critical events specify the formation of skeletal muscle: the expression of the myogenic transcription factor, XMyoD, and the secretion of bone morphogenetic protein (BMP) antagonists by the adjacent Spemann organizer. Inhibition of BMP signaling during early gastrula stages converts XMyoD protein into an instructive differentiation factor in the DLMZ. Yet, the intracellular signaling factors connecting BMP antagonism and activation of XMyoD remain unknown. Our data show that BMP antagonism induces the activity of mitogen-activated protein kinase (MAPK), and that the activity of MAPK is necessary for muscle-specific differentiation. Treatment of gastrula-stage DLMZ explants with MAPK pathway inhibitors ventralized mesoderm and prevented muscle differentiation. Expression of XMyoD in ventral mesoderm weakly induced muscle formation; however, the coexpression of a constitutively active MEK1 with XMyoD efficiently induced muscle differentiation. Activation of the MAPK pathway did not induce the transcription of XMyoD, but increased its protein levels and transcriptional activity. Thus, MAPK activation is subsequent to BMP antagonism, and participates in the dorsalization of mesoderm by converting the XMyoD protein into a potent differentiation factor.

Novel NEMO/IκB Kinase and NF-κB Target Genes at the Pre-B to Immature B Cell Transition

The IkappaB kinase (IKK) signaling complex is responsible for activating NF-kappaB-dependent gene expression programs. Even though NF-kappaB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-kappaB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cells versus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKgamma/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-kappaB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118, Pea-15/MAT1, CD82, CD40L, Wnt10a, Notch 1, R-ras, Rgs-16, PAC-1, ISG15, and CD36) and five repressed (CCR2, VpreB, lambda5, SLPI, and CMAP/Cystatin7) genes, respectively, were bona fide NF-kappaB targets by virtue of their response to a transdominant IkappaBalphaSR (super repressor). MyD118 and ISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-kappaB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Insulin-sensitive phospholipid signaling systems and glucose transport. Update II.

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidylcholine with subsequent increases in phosphatidic acid (PA) and diacylglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

Intracellular Signaling Factors—Enhanced Hepatic Nuclear Protein Binding to TTGGC Sequence in the Rat Regucalcin Gene Promoter: Involvement of Protein Phosphorylation

The transcriptional mechanism of regucalcin gene expression was determined using gel mobility shift assay with TTGGC oligonucleotide (II-b) which is located between position −523 and −506 in the promoter region, containing a nuclear factor I (NF1) consensus motif TTGGC(N)6CC. The mutation analysis in this motif showed that TTGGC sequence was a specific binding region of the nuclear protein in rat liver and the cloned rat hepatoma cells (H4-II-E). When liver nuclei were incubated with ATP (1 mM), the nuclear protein binding to TTGGC sequence was increased. This binding was also increased in the nuclei of H4-II-E cells cultured with 10% FBS. Such an increase was also seen by culture with vanadate (100 μM), a potent inhibitor of protein tyrosine phosphatase. Serum-enhanced nuclear protein binding to TTGGC sequence was decreased in the presence of TFP (10 μM), staurosporine (100 nM), genistein (10 μM), PD98059 (10 μM), or wortmannin (10 nM), which are inhibitors of various protein kinases. Treatment of a monoclonal phosphotyrosine antibody (4G10) caused an alteration in the TTGGC oligonucleotide–nuclear protein complex formation, indicating that tyrosine phosphorylation of nuclear protein is partly involved in the binding to TTGGC sequence. Moreover, when H4-II-E cells were cultured with FBS (10%), Bay K 8644 (5 μM), PMA (1 μM), or insulin (20 nM), the protein binding to TTGGC sequence in the nuclei was increased, while it was reduced in the cytoplasm, indicating a nuclear localization of the TTGGC sequence-binding protein. This study demonstrates that hepatic nuclear protein can specifically bind to the TTGGC sequence in rat regucalcin gene promoter region, and that this binding is enhanced by intracellular signaling factors which are partly mediated through protein phosphorylation.

Perturbation of myelination by activation of distinct signaling pathways: an in vitro study in a myelinating culture derived from fetal rat brain

An in vitro myelinating mouse-derived model system has been adapted and optimized for fetal rat brain. In these mixed brain cell (MBC) cultures, myelinogenesis was studied by examining the effect of signaling pathways that are involved in the timing of oligodendrocyte differentiation. When PMA, a protein kinase C (PKC) activator, was kept present during development, the early myelin protein, CNP, was expressed in oligodendrocytes as promptly as in control MBC cultures. In contrast, continuous activation of signaling pathways triggered by FGF-2 caused a delay in the expression of CNP. The expression of the late myelin proteins MBP and PLP in oligodendrocytes was impeded by both PMA- and FGF-2-treatment, and, as a consequence, also myelin formation. Surprisingly, the continuous presence of PDGF during development also prevented myelin formation, even though all myelin-specific proteins were significantly expressed. Taken together, the data indicate that this in vitro myelinating culture system represents an excellent system to study signaling events necessary for the onset of myelination. Moreover, the present results demonstrate that oligodendrocyte differentiation in the presence of neurons and astrocytes can be manipulated both by extracellular and intracellular signaling factors. Importantly, differentiation per se is not necessarily culminating into myelination.

Involvement of intracellular signaling factors in the serum-enhanced Ca2+-binding protein regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E)

The involvement of signaling factors, which are related to serum component, on the regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). H4-II-E cells were cultured for 2 or 6 h in a medium containing various reagents in the presence of serum (10% fetal bovine serum) after the subconfluent with 3-day-culture. The regucalcin mRNA expression was significantly increased by serum addition. This increase was clearly inhibited by the presence of EGTA (10(-3) M), A23187 (10(-6) M), trifluoperazine (10(-5) M), staurosporine (10(-7) M), or genistein (10(-5) M) with 6-h-culture, although the beta-actin mRNA expression was not altered by the reagents. Meanwhile, the regucalcin mRNA expression was significantly stimulated by the addition of Bay K 8644 (2.5 x 10(-6) M) in the presence of serum. This effect was also seen in the presence of genistein (10(-5) M). The present study suggests that the regucalcin mRNA expression is mediated through signaling pathways which are partly involved in Ca2+-dependent protein kinases and tyrosine kinase in H4-II-E hepatoma cells.

Involvement of intracellular signaling factors in the serum‐enhanced Ca2‐binding protein regucalcin mRNA expression in the cloned rat hepatoma cells (H4‐II‐E)

Involvement of intracellular signaling factors in the serum‐enhanced Ca2‐binding protein regucalcin mRNA expression in the cloned rat hepatoma cells (H4‐II‐E)

Insulin-Sensitive Phospholipid Signaling Systems and Glucose Transport: An Update

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidylcholine with subsequent increases in phosphatidic acid (PA) and diacylglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

Drugs acting on the intracellular signalling system

Cellular response to extracellular messages is a basic process to maintain and to support cell life. Several signalling molecules important as sites of therapeutic drug action are involved in the response. Recent studies on life sciences have elucidated molecular properties of intracellular signalling factors and mechanisms of cascading. Novel drugs acting on signalling molecules and possessing new sites and mechanisms of action have been found. This article summarizes the properties (subtypes, structures, functions) of signalling factors (receptors, ion channels, GTP binding proteins, second messenger-generating enzymes, second messenger-metabolizing enzymes, second messengers protein kinases, protein phosphatases) and lists in Tables A-H drugs that act on signalling molecules and which should find clinical use.