Mitogen-stimulated activation of the Na+/H+ antiporter does not regulate S6 phosphorylation or protein synthesis in murine thymocytes or Swiss 3T3 fibroblasts

The activation of protein synthesis by mitogens in quiescent (G0) mammalian cells is obligatory for progression from G0 through G1 to DNA synthesis in S phase. When the activation of the Na+/H+ antiporter which occurs in mitogen-stimulated Swiss 3T3 fibroblasts or murine fibroblasts is completely blocked by dimethylamiloride, there is little or no effect on the phosphorylation of the ribosomal protein S6 or the activation of protein synthesis assayed by [35S]methionine incorporation. Furthermore, the accumulation of the protein product of the activated c-myc gene is unaffected by dimethylamiloride in 3T3 fibroblasts. The data show that there is no requirement for activation of the Na+/H+ antiporter for the activation of S6 phosphorylation or protein synthesis by mitogens but do not preclude the possibility that activation of the antiporter is necessary for some other response(s) obligatory for DNA synthesis. These data are contrasted with previous reports for Chinese hamster lung fibroblasts that the increase in intracellular pH which results from activation of the Na+/H+ antiporter in bicarbonate-free media is necessary for S6 phosphorylation, protein synthesis, and hence, for subsequent DNA synthesis (Pouyssegur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939; Chambard, J.C., and Pouyssegur, J. (1986) Exp. Cell Res. 164, 282-294).     

Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 Activation

S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.     

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.     

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.     

TPA stimulates S6 phosphorylation but not protein synthesis in Ehrlich cells

Increased phosphorylation of ribosomal protein S6 has been extensively correlated with an increased rate of protein synthesis. We report here that under two separate conditions in Ehrlich cells an increase in the level of S6 phosphorylation does not result in any increase in the rate of protein synthesis. 1) In glutamine-deprived cells TPA stimulates S6 phosphorylation but has no effect on the rate of protein synthesis, 2) In cells deprived of serum growth factors, addition of serum stimulates both S6 phosphorylation and protein synthesis while TPA stimulates only S6 phosphorylation. These results show that increased phosphorylation of S6 is not sufficient to cause increased rates of protein synthesis, and suggest that additional factors may play a more direct role.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.     

Cyclic AMP increasing agents rapidly stimulate vimentin phosphorylation in quiescent cultures of Swiss 3T3 cells

The results presented here demonstrate that an elevation in the cellular levels of cyclic AMP (cAMP) increases the phosphorylation of an Mr = 58,000 cellular protein in quiescent cultures of Swiss 3T3 cells. The enhancement of 32Pi incorporation into the Mr 58,000 cellular protein was detected as early as 1 min and reached a maximum after 20 min of treatment. The role of cAMP in the phosphorylation of Mr = 58,000 protein is substantiated by the following lines of evidence: a) a variety of agents that cause cAMP accumulation in 3T3 cells, including cholera toxin, 5'-N-ethylcarboxamideadenosine (NECA), PGE1, and 3-isobutyl-1-methyl-xanthine (IBMX) increased the phosphorylation of the same Mr 58,000 cellular protein as demonstrated by peptide mapping; b) inhibitors of cyclic nucleotide phosphodiesterase potentiated the ability of low concentrations of the adenylate cyclase activators NECA, PGE1, and forskolin to increase Mr 58,000 phosphorylation; and c) permeable derivatives of cAMP such as 8BrcAMP were also effective and specific in promoting Mr 58,000 phosphorylation. Detergent extraction, immunoblotting, and immunoprecipitation identified the Mr = 58,000 phosphoprotein as vimentin, the main protein subunit of the intermediate filaments of mesenchymal cells including Swiss 3T3 cells. Studies with intact 3T3 cells revealed that an increase in the intracellular level of cAMP induced a marked redistribution and collapse of the intermediate filaments. These results raise the possibility that an intact intermediate filament network may restrict the reinitiation of DNA synthesis.     

EGF, PGF2 alpha and insulin induce the phosphorylation of identical S6 peptides in swiss mouse 3T3 cells: effect of cAMP on early sites of phosphorylation

Epidermal growth factor (10(-9)M), prostaglandin (8.5 X 10(-7)M), F2 alpha, and insulin (10(-9)M), each of which only leads to a partial phosphorylation of 40S ribosomal protein S6, generate the same first eight phosphopeptides induced by 10% serum, suggesting all three activate a common regulatory pathway for the phosphorylation of S6. Added together, they induce almost maximal S6 phosphorylation and a phosphopeptide pattern nearly equivalent to that of serum. Unlike the agents above, 8-Br-cAMP or PGE1 has no significant effect on protein synthesis, but does induce a small increase in S6 phosphorylation. Surprisingly, the three peptides that become phosphorylated are identical with insulin-induced phosphopeptides 10b, 11, and 9, based on either comigration, limited acid hydrolysis, or V8 protease digestion. Incubation of 40S subunits with cAMP-dependent protein kinase induces the phosphorylation of these same three phosphopeptides. The in vitro and in vivo studies described here raise the possibility that cAMP could, in part, be responsible for mediating the phosphorylation of S6 during the mitogenic response.     

Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.     

Potassium transport and content during G1 and S phase following serum stimulation of 3T3 cells.

The effect of serum stimulation on unidirectional and net K flux and their relationship to the initiation of DNA synthesis has been investigated in mouse 3T3 fibroblasts. Stimulation of quiescent 3T3 cells with 20% serum results in the initiation of S phase approximately ten hours after serum addition. During transition from G₁ to S phase distinct changes in K transport and cellular K content occur. Total unidirectional K influx undergoes an immediate 2-fold increase upon serum addition, an observation in qualitative agreement with previous results (Rozengurt and Heppel, 1975). This total increase in unidirectional K influx represents a proportional increase in the active, ouabain sensitive component and the K-K exchange component. The initial increase in total flux is followed by a gradual decline over a 16-hour period to levels approaching those of quiescent cells. Following the initial increase in unidirectional K influx is an approximately 75% increase in cell K on a per milligram protein basis or a 40% increase on a per volume basis. This increase peaks at four to five hours and then declines to initial levels at 10 to 14 hours. Populations of quiescent cells given 20% serum plus 0.5 mM ouabain simultaneously are totally blocked from entering S phase, as determined by the appearance of ³H-thymidine labeled nuclei. However, if the ouabain is removed after six hours these cells then undergo the same changes in unidirectional K influx and content as serum stimulated cells with entrance into S phase retarded by five to six hours. If ouabain is added to serum stimulated cells at six hours, after the increase in K transport and K content have occurred, entrance into S phase is not entirely blocked. In cells stimulated with serum and 0.5 mM dBcAMP plus 1 mM theophylline simultaneously, entrance into S phase is greatly reduced as compared to serum stimulation only. However, the early and late changes in K flux and K content are not substantially altered. This indicates that the K transport events associated with G₁ and early S phase are not directly regulated by changes in cAMP levels which follow serum stimulation.     

Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3.

Intracellular signaling pathways that are involved in protection of vascular smooth muscle cells (VSMC) from apoptosis remain poorly understood. This study examines the effect of activators of cAMP/cGMP signaling on apoptosis in non-transfected VSMC and in VSMC transfected with c-myc (VSMC-MYC) or with its functional analogue, E1A-adenoviral protein (VSMC-E1A). Serum-deprived VSMC-E1A exhibited the highest apoptosis measured as the content of chromatin and low molecular weight DNA fragments, phosphatidylserine content in the outer surface of plasma membrane and caspase-3 activity (ten-, five-, four- and tenfold increase after 6 h of serum withdrawal, respectively). In VSMC-E1A, the addition of an activator of adenylate cyclase, forskolin, abolished chromatin cleavage, DNA laddering, caspase-3 activation and the appearance of morphologically-defined apoptotic cells triggered by 6 h of serum deprivation. In non-transfected VSMC and in VSMC-MYC, 6 h serum deprivation led to approximately six- and threefold activation of chromatin cleavage, respectively, that was also blocked by forskolin. In VSMC-E1A, inhibition of apoptosis was observed with other activators of cAMP signaling (cholera toxin, isoproterenol, adenosine, 8-Br-cAMP), whereas 6 h incubation with modulators of cGMP signaling (8-Br-cGMP, nitroprusside, atrial natriuretic peptide, L-NAME) did not affect the development of apoptotic machinery. The antiapoptotic effect of forskolin was abolished in 24 h of serum deprivation that was accompanied by normalization of intracellular cAMP content and protein kinase A (PKA) activity. Protection of VSMC-E1A from apoptosis by forskolin was blunted by PKA inhibitors (H-89 and KT5720), whereas transfection of cells with PKA catalytic subunit attenuated apoptosis triggered by serum withdrawal. The protection of VSMC-E1A by forskolin from apoptosis was insensitive to modulators of cytoskeleton assembly (cytochalasin B, colchicine). Neither acute (30 min) nor chronic (24 h) exposure of VSMC to forskolin modified basal and serum-induced phosphorylation of the MAP kinase ERK1/2. Thus, our results show that activation of cAMP signaling delays the development of apoptosis in serum-deprived VSMC at a site upstream of caspase-3 via activation of PKA and independently of cAMP-induced reorganization of the cytoskeleton network and the ERK1/2-terminated MAPK signaling cascade.     

Cyclic 3'',5''-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signaling response

Refinement of a perfusion technique permitted the simultaneous measurement of cAMP-elicited [3H]cAMP secretion and intracellular [3H]cAMP levels in sensitive D. discoideum amoebae. These data were compared with measurements of the rate of [32P]cAMP synthesis by extracts of amoebae sonicated at different times during the cAMP signaling response. cAMP stimulation of intact cells led to a transient activation of adenylate cyclase, which was blocked if 10(-4) M NaN3 was added with the stimulus. During responses elicited by 10(-6) M cAMP, 10(-8) M cAMP, and an increment in cAMP from 10(-8) M to 10(-7) M, the rate of cAMP secretion was proportional to the intracellular cAMP concentration. Removal of a 10(-6) M cAMP stimulus 2 min after the initiation of the response led to a precipitous decline in intracellular cAMP. This decline was more rapid than could be accounted for by secretion alone, suggesting intracellular phosphodiesterase destruction of newly synthesized cAMP. Employing these data and a simple rate equation, estimates of the time-course of the transient activation of adenylate cyclase and the rate constants for cAMP secretion and intracellular phosphodiesterase activity were obtained. The calculated rate of cAMP synthesis rose for approximately 1 to 2 min, peaked, and declined to approach prestimulus levels after 3 to 4 min. This time-course agreed qualitatively with direct measurements of the time-course of activation, indicating that the activation of adenylate cyclase is a major in determining the time-course of the cAMP secretion response.     

Effects of cyclic AMP and fluoride on phosphorylation of ribosomal protein S6 and on protein synthesis in rabbit reticulocytes

The effects of N6,O2-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) and sodium fluoride on the phosphorylation of ribosomal proteins S6 and on protein synthesis were examined. Rabbit reticulocytes were incubated in a nutritional medium containing 32Pi in the presence and absence of Bt2cAMP (1mM) and 3-isobutyl-1-methyl-xanthine (1mM). In the control cells, four phosphorylated derivatives of S6 were observed, with most of the radioactivity in the monophosphorylated form. Upon addition of cyclic nucleotide, a twofold increase in the phosphorylation of ribosomal protein S6 was observed. This was accompanied by an increase of radioactive phosphate in the diphosphorylated derivative. No alteration in protein synthesis was observed upon addition of cAMP and analogues of cAMP in conjunction with 3-isobutyl-1-methyl-xanthine or theophylline. The effects of sodium fluoride on phosphorylation of S6 and on protein synthesis were examined also. At 5 mM sodium fluoride, protein synthesis was inhibited by 85%. A 2.5-fold increase in the phosphorylation of ribosomal protein S6 was observed with an accumulation of 32Pi in the diphosphorylated, triphosphorylated and tetraphosphorylated derivatives. Inhibition of protein synthesis coincided with an increase in the more highly phosphorylated derivatives, whereas an increase of radioactive phosphate in the diphosphorylated derivative could not be correlated with an alteration in globin synthesis.     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells

We have previously shown that the vasoconstrictive peptide angiotensin II (ANG II) is a hypertrophic agent for human coronary artery smooth muscle cells (cSMCs), which suggests that it plays a role in vascular wall thickening. The present study investigated the intracellular signal transduction pathways involved in the growth response of cSMCs to ANG II. The stimulation of protein synthesis by ANG II in cSMCs was blocked by the immunosuppressant rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway that includes the 70-kDa S6 kinase (p70(S6k)) and plays a key role in cell growth. The inhibitory effect of rapamycin was reversed by a molar excess of FK506; this indicates that both agents act through the common 12-kDa immunophilin FK506-binding protein. ANG II caused a rapid and sustained activation of p70(S6k) activity that paralleled its phosphorylation, and both processes were blocked by rapamycin. In addition, both of the phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 abolished the ANG II-induced increase in protein synthesis, and wortmannin also blocked p70(S6k) phosphorylation. Furthermore, ANG II triggered dissociation of the translation initiation factor, eukaryotic initiation factor-4E, from its regulatory binding protein 4E-BP1, which was also inhibited by rapamycin and wortmannin. In conclusion, we have shown that ANG II activates components of the rapamycin-sensitive mTOR signaling pathway in human cSMCs and involves activation of phosphatidylinositol 3-kinase, p70(S6k), and eukaryotic initiation factor-4E, which leads to activation of protein synthesis. These signaling mechanisms may mediate the growth-promoting effect of ANG II in human cSMCs.     

Hypoxia-induced remodelling of PDE4 isoform expression and cAMP handling in human pulmonary artery smooth muscle cells

Human pulmonary artery smooth muscle cells (hPASM cells) express PDE4A10, PDE4A11, PDE4B2, PDE4C and PDE4D5 isoforms. Hypoxia causes a transient up-regulation of PDE4B2 that reaches a maximum after 7 days and sustained up-regulation of PDE4A10/11 and PDE4D5 over 14 days in hypoxia. Seven days in hypoxia increases both intracellular cAMP levels, protein kinase A (PKA) activity and activated, phosphorylated extracellular signal regulated kinase (pERK) but does not alter either PKA isoform expression or total cAMP phosphodiesterase-4 (PDE4) activity or cAMP phosphodiesterase-3 (PDE3) activity. Both the cyclooxygenase inhibitor, indomethacin and the ERK inhibitors, UO126 and PD980589 reverse the hypoxia-induced increase in intracellular cAMP levels back to those seen in normoxic hPASM cells. Challenge of normoxic hPASM cells with prostaglandin E(2) (PGE(2)) elevates cAMP to levels comparable to those seen in hypoxic cells but fails to increase intracellular cAMP levels in hypoxic hPASM cells. The adenylyl cyclase activator, forskolin increases cAMP levels in both normoxic and hypoxic hPASM cells to comparable elevated levels. Challenge of hypoxic hPASM cells with indomethacin attenuates total PDE4 activity whilst challenge with UO126 increases total PDE4 activity. We propose that the hypoxia-induced activation of ERK initiates a phospholipase A(2)/COX-driven autocrine effect whereupon PGE(2) is generated, causing the activation of adenylyl cyclase and increase in intracellular cAMP. Despite the hypoxia-induced increases in the expression of PDE4A10/11, PDE4B2 and PDE4D5 and activation of certain of these long PDE4 isoforms through PKA phosphorylation, we suggest that the failure to see any overall increase in PDE4 activity is due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. Such hypoxia-induced increase in expression of PDE4 isoforms known to interact with certain signalling scaffold proteins may result in alterations in compartmentalised cAMP signalling. The hypoxia-induced increase in cAMP may represent a compensatory protective mechanism against hypoxia-induced mitogens such as endothelin-1 and serotonin.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.