Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells

Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P(1)-S1P(5)). Of these receptors, S1P(1), S1P(2), and S1P(3) are ubiquitously expressed in adult mice, while S1P(4) and S1P(5) are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P(2) mRNA was abundantly expressed in F9 EC cells, but little S1P(1) and no S1P(3), S1P(4), or S1P(5) mRNA was detectable. However, S1P(1) mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P(1) indicated that increased S1P(1) expression is required for PrE differentiation. Thus, S1P(1) may play an important function in PrE differentiation that is not substituted for by S1P(2).     

MEKK4 mediates differentiation in response to retinoic acid via activation of c-Jun N-terminal kinase in rat embryonal carcinoma P19 cells

Differentiation of P19 embryonal carcinoma cells in response to the morphogen retinoic acid is regulated by Galpha(12/13) and is associated with activation of c-Jun N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun N-terminal kinase was investigated in P19 cells. P19 clones stably expressing constitutively active and dominant negative mutants of MEKK1 and MEKK4 were created and characterized. Expression of the constitutively active form of either MEKK1 or MEKK4 mimicked the action of retinoic acid, inducing these embryonal carcinoma cells to primitive endoderm. Expression of the dominant negative form of MEKK1 had no influence on the ability of retinoic acid to induce either JNK activation or primitive endoderm formation in P19 stem cells. Expression of the dominant negative form of MEKK4, in contrast, effectively blocks both morphogen-induced activation of JNK and cellular differentiation. These data identify MEKK4 as upstream of c-Jun N-terminal kinase in the pathway mediating differentiation of P19 stem cells to primitive endoderm.     

Occurrence in the archaebacterium Sulfolobus solfataricus of a ribosomal protein complex corresponding to Escherichia coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)2.P0

Two-dimensional electrophoresis of total protein from 50 S ribosomal subunits of the archaebacterium Sulfolobus solfataricus demonstrated a complex between two proteins that was stable in 6 M urea, but dissociable in detergent or below pH 5.5. The proteins, numbered L1 and L10 according to their electrophoretic mobilities, corresponded to Escherichia coli ribosomal proteins L10 and L7/L12, respectively. The members of the complex were therefore designated Sso L10e and Sso L12e. Sso L12e had other properties in common with E. coli L7/L12: low molecular weight, relative acidity, selective release from the ribosome by high salt/ethanol, and dimeric structure. The Sso L12e.Sso L10e complex was isolated by gel filtration of total 50 S proteins in 4 M urea. The stoichiometry of the components was approximately four copies of Sso L12e to one copy of Sso L10e. The occurrence in an archaebacterium of a complex of acidic ribosomal proteins similar to E. coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)/.P0 strongly supports the concept that this element of quaternary structure is a major conserved feature of the ribosome and reaffirms its importance in the translocation step of protein synthesis.     

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE₂ formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β).     

Galpha(i2) enhances insulin signaling via suppression of protein-tyrosine phosphatase 1B

Suppression of the expression of the heterotrimeric G-protein Galpha(i2) in vivo has been shown to provoke insulin resistance, whereas enhanced insulin signaling is observed when Galpha(i2) is overexpressed in vivo. The basis for Galpha(i2) regulation of insulin signaling was explored in transgenic mice with targeted expression of the GTPase-deficient, constitutively active Q205L Galpha(i2) in fat and skeletal muscle. Phosphorylation of insulin receptor and IRS-1 in response to insulin challenge in vivo was markedly amplified in fat and skeletal muscle expressing Q205L Galpha(i2). The expression and activity of the protein-tyrosine phosphatase 1B (PTP1B), but not protein-tyrosine phosphatases SHP-1, SHP-2, and LAR, were constitutively decreased in tissues expressing the Q205L Galpha(i2), providing a direct linkage between insulin signaling and Galpha(i2). The loss of PTP1B expression may explain, in part, the loss of PTP1B activity in the iQ205L transgenic mice. Activation of Galpha(i2) in mouse adipocytes with lysophosphatidic acid was shown to decrease PTP1B activity, whereas pertussis toxin inactivates Galpha(i2), blocks lysophosphatidic acid-stimulated inhibition of PTP1B activity, and blocks tonic suppression of PTP1B activity by Galpha(i2). Elevation of intracellular cAMP in fat cells is shown to increase PTP1B activity, whereas either depression of cAMP levels or direct activation of Galpha(i2) suppresses PTP1B. These data provide the first molecular basis for the interplay between Galpha(i2) and insulin signaling, i.e. activation of Galpha(i2) can suppress both the expression and activity of PTP1B in insulin-sensitive tissues.     

LncRNA-uc.40 silence promotes P19 embryonic cells differentiation to cardiomyocyte via the PBX1 gene

Uc.40 is a long noncoding RNA that is highly conserved among different species, although its function is unknown. It is highly expressed in abnormal human embryonic heart. We previously reported that overexpression of uc.40 promoted apoptosis and inhibited proliferation of P19 cells, and downregulated PBX1, which was identified as a potential target gene of uc.40. The current study evaluated the effects of uc40-siRNA-44 (siRNA against uc.40) on the differentiation, proliferation, apoptosis, and mitochondrial function in P19 cells, and investigated the relationship between uc.40 and PBX1 in cardiomyocytes. The uc.40 silencing expression was confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Observation of morphological changes in transfected P19 cells during different stages of differentiation revealed that uc40-siRNA-44 increased the number of cardiomyocyes. There was no significant difference in the morphology or time of differentiation between the uc40-siRNA-44 group and the control group. uc40-siRNA-44 significantly promoted proliferation of P19 cells and inhibited serum starvation-induced apoptosis. There was no significant difference in mitochondrial DNA copy number or cellular ATP level between the two groups, and ROS levels were significantly decreased in uc40-siRNA-44-transfected cells. The levels of PBX1 and myocardial markers of differentiation were examined in transfected P19 cells; uc40-siRNA-44 downregulated myocardial markers and upregulated PBX1 expression. These results suggest that uc.40 may play an important role during the differentiation of P19 cells by regulation of PBX1 to promote proliferation and inhibit apoptosis. These studies provide a foundation for further study of uc.40/PBX1 in cardiac development.     

Activation of rat frizzled-1 promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via pathways that require Galpha(q) and Galpha(o) function

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either Galpha(o) or Galpha(q) via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.     

Variation in intracellular P1P4-bis(5''-adenosyl) tetraphosphate (Ap4A) in virus-infected cells.

The effect of virus infection on the intracellular concentration of the proposed stress alarmone P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) has been examined in Vero cells. Compared with exposure to 0.8 mM-Cd2+, which causes a 30-fold increase in Ap4A, infection with simian virus 40 and poliovirus causes only a 2-fold increase, whereas herpes simplex virus type 1 results in a decrease in Ap4A during the course of the infection.     

Maitotoxin induces a calcium-dependent membrane depolarization in GH4C1 pituitary cells via activation of type L voltage-dependent calcium channels.

Maitotoxin (MTX) is a water-soluble polyether, isolated from the marine dinoflagellate Gambierdiscus toxicus, that stimulates hormone release and Ca2+ influx. We have investigated the action by which MTX induces Ca2+ influx and stimulates prolactin (PRL) release from GH4C1 rat pituitary cells. PRL release elicited by MTX is abolished in a concentration-dependent manner by nimodipine, a dihydropyridine (DHP) antagonist of type L voltage-dependent calcium channels (L-VDCC), indicating that MTX-enhanced PRL release occurs via activation of type L-VDCC. As an initial approach to determine whether MTX interacts directly with VDCC, we examined whether MTX affects the binding of [3H]PN 200-110, a DHP class antagonist, in intact GH4C1 cells. MTX increased the Bmax of [3H]PN 200-110 binding to intact GH4C1 cells from 4.6 +/- 0.03 to 12.5 +/- 2.2 fmol/10(6) cells, without changing the Kd. This indicates that MTX does not bind to the DHP site, but rather suggests that MTX may have an allosteric interaction with the DHP binding site. The effect of MTX on DHP binding was largely (65%) calcium-dependent. We next examined whether MTX alters the membrane potential of GH4C1 cells using the potential sensitive fluorescent dye bisoxonol. Addition of 100 ng/ml MTX to GH4C1 cells caused a membrane depolarization within 2.5 min which reached a plateau at 5 min. The MTX-induced depolarization was not prevented by substitution of impermeant choline ions for Na+. It was similarly unaffected by K+ channel blockers or by depleting the K+ chemical concentration gradient with gramicidin, a monovalent cation pore-forming agent. By contrast, low extracellular Ca2+ totally abolished the depolarization response, and nimodipine at 100 nM substantially reduced the MTX-induced membrane depolarization. These results indicate that the predominant effect of MTX on depolarization is Ca2+ influx through L-VDCC. Taken together, our results indicate that MTX-enhanced PRL release occurs exclusively via activation of type L-VDCC in GH4C1 cells. We suggest that MTX induces an initial slow calcium conductance, possibly via an allosteric interaction with a component of the VDCC complex, which, in turn, initiates a positive feedback mechanism involving calcium-dependent membrane depolarization and voltage-dependent activation of calcium channels.     

D-Ala2, (F5) Phe4]-dynorphin 1-13-NH2 (DAFPHEDYN): a potent analog of dynorphin 1-13

Intracerebroventricular administration of the dynorphin analog, [D-Ala2,(F5)Phe4]-dynorphin 1-13-NH2 (DAFPHEDYN) in rats produced diuresis and profound analgesia. Both effects were antagonized by central administration of naltrexone or naloxone. Intravenous administration of 10, 25, and 50 mg/kg of DAFPHEDYN failed to induce diuresis. The increased potency of DAFPHEDYN was apparent from the failure of an equal dose of the parent compound (dynorphin 1-13) to produce diuresis and the failure of [D-Ala2]-dynorphin 1-13-NH2 to produce analgesia. Radioligand binding studies indicated the DAFPHEDYN retains the same degree of kappa selectivity as the parent compound (dynorphin 1-13) though a drop in affinity occurred. DAFPHEDYN may be of significant interest because it retains the essential pharmacology of the parent compound and exhibits marked in vivo potency.     

Activation of P2Y1 and P2Y2 receptors induces chloride secretion via calcium-activated chloride channels in kidney inner medullary collecting duct cells

Dysregulation of urinary sodium chloride (NaCl) excretion can result in extracellular fluid (ECF) volume expansion and hypertension. Recent studies demonstrated that urinary nucleotide excretion increases in mice ingesting a high-salt diet and that these increases in extracellular nucleotides can signal through P2Y(2) receptors in the kidney collecting duct to inhibit epithelial Na(+) channels (ENaC). However, under conditions of ECF volume expansion brought about by high-dietary salt intake, ENaC activity should already be suppressed. We hypothesized that alternative pathways exist by which extracellular nucleotides control renal NaCl excretion. We used an inner medullary collecting duct (mIMCD-K2) cell line in an Ussing chamber system as a model to study additional ion transport pathways that are regulated by extracellular nucleotides. When ENaC was inhibited, the addition of adenosine triphosphate (ATP) to the basal side of cell sheets activated both P2Y(1) and P2Y(2) receptors, inducing a transient increase in short-circuit current (I(sc)); addition of ATP to the apical side activated only P2Y(2) receptors, inducing first a transient and then a sustained increase in I(sc). The ATP-induced increases in I(sc) were blocked by pretreatment with a phospholipase C (PLC) inhibitor, a calcium (Ca(2+)) chelator, or Ca(2+)-activated Cl(-) channel (CACC) inhibitors, suggesting that ATP signals through both PLC and intracellular Ca(2+) to activate CACC. We propose that P2Y(1) and P2Y(2) receptors operate in tandem in IMCD cells to provide an adaptive mechanism for enhancing urinary NaCl excretion in the setting of high-dietary NaCl intake.     

Early increase in histone H1(0) mRNA during differentiation of F9 cells to parietal endoderm.

We have isolated and characterized cDNA clones coding for the H1 histone subtype H1(0) in mouse teratocarcinoma cells. The mRNA is 2100 nt long and contains a coding sequence which is highly related to that of the human H1(0) gene. Using this cDNA as a probe, we have shown that, in comparison to undifferentiated F9 cells, differentiated F9 teratocarcinoma cells contain large amounts of H1(0) mRNA. This increase takes place very early during differentiation and does not correlate with changes in the rate of cell division. This indicates that the accumulation of H1(0) mRNA is not the result of reduced proliferation. Most likely on the contrary, the increase in the amount of H1(0) and the resulting effects on the formation of high order chromatin structures are parts of the differentiation program induced in F9 cells.     

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, induces apoptosis of endothelial cells

Smoking causes endothelial cell (EC) injury; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury are understood. The nitrosated derivative of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been implicated in the carcinogenic effects of tobacco; however, the effects of NNK on the cardiovascular system are largely unknown. NNK binds to beta1- and beta2-adrenergic receptors. Because beta-adrenergic receptor activation causes arachidonic acid (AA) release and cellular injury, we postulated that NNK causes EC injury by a mechanism that involves beta-adrenergic-mediated release of AA. NNK stimulated [3H]AA release from ECs, and this effect was mediated by both beta1- and beta2-adrenergic receptors because pretreatment with atenolol or ICI 118,551 inhibited the response. NNK also induced EC apoptosis, as measured by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and annexin V staining. NNK-mediated apoptosis was attenuated by pretreatment with atenolol or ICI 118,551. Furthermore, depletion of cellular AA by incubation with eicosapentaenoic acid abolished the apoptotic effect of NNK. These data suggest that NNK causes EC apoptosis by a mechanism that involves beta1- and beta2-adrenergic receptor-mediated release of AA.     

Coupling of the muscarinic m2 receptor to G protein-activated K(+) channels via Galpha(z) and a receptor-Galpha(z) fusion protein. Fusion between the receptor and Galpha(z) eliminates catalytic (collision) coupling

G protein-activated K(+) channel (GIRK), which is activated by the G(betagamma) subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K(+) current, I(ACh), via the endogenous pertussis toxin (PTX)-sensitive G(i/o) proteins. Activation of I(ACh) was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the alpha subunit of the PTX-insensitive G protein G(z), Galpha(z), induced a slowly activating PTX-insensitive I(ACh), whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R x Galpha(z) fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Galpha(z), part of I(ACh) was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Galpha(z) but also the nontethered G(i/o) proteins. After PTX treatment, the speed of activation of the m2R x Galpha(z)-mediated response did not depend on the expression level of m2R x Galpha(z) and was faster than when m2R and Galpha(z) were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Galpha strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system.