Endothelin—1 promoted proliferation of vascular smooth muscle cell through pathway of extracellular signal—regulated kinase and cyclin D1

目的:探讨内皮素-1是否通过细胞周期蛋白质D1与细胞外调节蛋白激酶通路促进人脐动脉平滑肌细胞增殖。方法:采用MTT法观察ET-1和PD98059对人脐动脉平滑肌细胞生长的作用;[~3H]TdR法观察对细胞DNA合成的作用;流式细胞仪法观察对细胞增殖周期的影响;蛋白质印迹法观察对细胞外调节蛋白激酶和细胞周期蛋白质D1表达的影响。结果:首先,同没有ET-1组和PD98059组比较,ET-1促进平滑肌细胞增殖(P<0.05)。PD98059抑制ET-1诱导的血管平滑肌细胞增殖。第二,与没有ET-1组比较,ET-1促进平滑肌细胞DNA合成(P<0.05)。第三,ET-1促进平滑肌细胞增殖周期从G_0/G_1期向S期的转变,与没有ET-1组比较,G_0/G_1期细胞百分比明显减少,S期细胞百分比明显增加(P<0.05)。第四,ET-1增加细胞外信号调节性激酶的磷酸化水平和细胞周期蛋白质D1的蛋白表达,ERK的抑制剂可以抑制细胞外信号调节性激酶的磷酸化水平和细胞周期蛋白质D1的蛋白表达,与没有ET-1组比较,磷酸化-ERK和细胞周期蛋白质D1表达明显增强,对非磷酸化ERK表达没有影响。结论:内皮素-1可以通过细胞周期调节素D1与细胞外信号调节性激酶通路促进平滑肌细胞增殖。     

Mechanisms underlying lysophosphatidylcholine-induced potentiation of vascular contractions in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat aorta

BACKGROUND AND PURPOSE: The effect of lysophosphatidylcholine (LPC) on aortic contractions in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetic model, was studied. EXPERIMENTAL APPROACH: Using OLETF rats and control (Long Evans Tokushima Otsuka (LETO)) rats, the effects of LPC on the contractions induced by high-K(+) (10-40 mM), UK14,304 (10 approximately 100 nM; a selective alpha(2)-adrenoceptor agonist) and sodium orthovanadate (SOV; 10 microM approximately 3 mM) in endothelium-denuded aortae were compared. Aortic ERK activity and the mRNA expression for GPR4 (a putative LPC receptor) were also measured. KEY RESULTS: OLETF rats exhibited (vs. age-matched LETO rats): (1) greater potentiation of high-K(+)-induced contraction by 10 microM LPC - a potentiation attenuated by 10 microM genistein, protein tyrosine kinase (PTK) inhibitor, (2) greater potentiation of UK14,304 (10 approximately 100 nM)-induced contractions by LPC (1 microM approximately 10 microM) - a potentiation attenuated by 10 microM genistein, 50 microM tyrphostin A23 (PTK inhibitor) or 10 microM PD98059 (MEK 1/2 inhibitor), (3) greater basal and LPC (1 microM)-induced ERK activities, (4) greater basal and 100 nM UK14,304-stimulated ERK2 activities in both the absence and presence of 10 microM LPC, (5) greater SOV (10 microM approximately 3 mM)-induced contractions, (6) greater potentiation of SOV-induced contractions by 10 microM LPC - a potentiation suppressed by 10 microM PD98059 or 10 microM genistein, (7) upregulation of GPR4 mRNA. CONCLUSIONS AND IMPLICATIONS: These results suggest that the LPC-induced potentiation of contractions in the OLETF rat aorta may be attributable to increased PTKs or ERK activity and/or to receptor upregulation.     

Contribution of extracellular signal-regulated kinases to the IL-1-induced growth inhibition of human melanoma cells A375

The role of ERK1/2 in the IL-1-induced growth inhibition was investigated using human melanoma A375-6 cells. A selective inhibitor of ERK1/2 pathway, PD98059 and a selective inhibitor of p38MAPK, SB203580 each alone significantly reversed the IL-1-induced growth inhibition of A375-6 cells. Co-treatment with PD98059 and SB203580 completely reversed the IL-1-induced growth inhibition. ERK1/2 was constitutively activated in A375-6 cells, and IL-1 further augmented ERK activation. Antiproliferative effect of IL-1 was attenuated by the expression of dominant negative form of ERK2. IL-1 induced cell cycle arrest in G(0)/G(1) phase, expression of p21 and p27 proteins, and down-regulation of cyclin D/cyclin-dependent kinase (CDK) 2 and CDK4 activities. These effects of IL-1 were reversed by PD98059. PD98059 also reversed the IL-1-induced hypophosphorylation of RB protein (pRB) and down-regulation of E2F activity. These findings demonstrate that ERK1/2 contribute to the IL-1-induced growth inhibition through induction of CDK inhibitors, down-regulation of CDK activity, pRB phosphorylation and E2F activity.     

Activation of cannabinoid CB2 receptor negatively regulates IL-12p40 production in murine macrophages: role of IL-10 and ERK1/2 kinase signaling

1 Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs for the treatment of many disease conditions. In the present study, we investigated the effects of the synthetic CB(2) ligand, JWH-133 on the production of interleukins (ILs), IL-12 and IL-10 by lipopolyssacharide (LPS) or Theiler's virus (TMEV)-activated macrophages. 2 JWH-133 evoked a concentration-related inhibition (10 nM-5 microM) of LPS/IFN-gamma induced IL-12p40 release. The effect of JWH-133 (100 nM) was significantly blocked by the CB2 antagonist SR-144528 (1 microM). Macrophages infected with TMEV increased IL-12p40 production and activation of CB2 receptors by JWH-133 (100 nM) inhibited it. 3 The inhibitory effect of JWH-133 (100 nM) on IL-12p40 production may involve extracellular-regulated kinase (ERK1/2) signaling: (i) JWH-133 induced a greater and sustained activation of ERK1/2 kinase in comparison with the level of activation observed following LPS; (ii) the inhibition of ERK1/2 by the specific inhibitor PD98059 increased LPS-induced IL-12p40 production in the presence or absence of JWH-133 suggesting a negative regulation of ERK pathway on IL-12p40 biosynthesis. 4 Activation of CB2 receptors by JWH-133 (10 nM-5 microM) enhanced IL-10 release by LPS/IFN-gamma-activated macrophages and addition of SR144558 (1 microM) totally blocked the effect of JWH (100 nM). 5 Inhibition of ERK by PD98059 significantly suppressed IL-10 production by LPS-activated macrophages. Endogenous IL-10 plays a modulatory role in IL-12 production. Blocking IL-10 with neutralizing antibody resulted in increased IL-12p40 secretion by LPS-activated macrophages in the absence or presence of JWH-133. In contrast, the addition of exogenous mIL-10 reduced the secretion of IL-12p40 in response to LPS.     

PD98059 triggers G1 arrest and apoptosis in human leukemic U937 cells through downregulation of Akt signal pathway

MEK/ERK pathways are frequently activated in acute myelogenous leukemia, and this signal pathway's inhibitor has made it an interesting candidate for cancer chemotherapy. Little is known, however, about the effects of cellular and molecular mechanisms on human leukemic U937 cells. In the present study, we found that treatment with PD98059 significantly arrests the G1 phase through up-regulation of cyclin-dependent kinase (Cdk) inhibitor, and produces morphological features of apoptosis in U937 cells, which were associated with poly(ADP-ribose)polymerase (PARP) cleavage and PLC-gamma1 degradation. PD98059 also decreased the Cdk-2, Cdk-4, cyclin D1, and cyclin E expression, and increased high levels of the mitotic inhibitors p16(INIa), p21(Waf1), and p27(Kip1). Also, Bcl-2's overexpression and a caspase-3 inhibitor z-DEVD-fmk significantly attenuated PD98059-induced apoptosis through the down-regulation of caspase-3 activity, but did not attenuate G1 phase arrest. Moreover, PD98059 down-regulated Akt phosphorylation and produced a synergy effect of apoptosis with LY294002 co-treatment. Thus, our results imply that PD98059-induced apoptosis is significantly involved in down-regulation of Bcl-2, caspase-3 activity, the Akt pathway, and some of the biological functions in U937 cells.     

D(4) dopamine receptor differentially regulates Akt/nuclear factor-kappa b and extracellular signal-regulated kinase pathways in D(4)MN9D cells

The present study was designed to investigate the role of D(4) dopamine receptors in regulating the Akt/nuclear factor-kappa B (NF-kappa B) and extracellular signal-regulated kinase (ERK) signaling pathways. The D(4) dopamine receptor agonist PD168077 induced time- and dose-dependent activation of Akt and ERK in D(4)MN9D cells that stably express D(4) dopamine receptors. Maximal Akt and ERK stimulation was achieved at 1 microM PD168077. The agonist-mediated stimulations of Akt and ERK were abolished when cells were preincubated with 50 ng/ml PTX or with 1 microM L745,870, a D(4) dopamine receptor antagonist, indicating that activation of the Akt or ERK pathways is mediated by D(4) dopamine receptors and require a pertussis toxin-sensitive G protein. We also detected a time- and dose-dependent activation of NF-kappa B. Activation of NF-kappa B by 1 microM PD168077 was attenuated in D(4)MN9D cells that were transfected with a kinase-deficient Akt but not in cells transfected with a dominant negative Ras (N17Ras), suggesting that NF-kappa B activation requires Akt but is independent of Ras. In contrast, the transfection of N17Ras into D(4)MN9D cells blunted D(4) dopamine receptor-mediated ERK activation, indicating a Ras-dependent mechanism. Moreover, PP2 (20 nM), an inhibitor of Src, blocked D(4) receptor-mediated SHC phosphorylation and ERK activation. In contrast, transfection of a kinase-dead Akt did not alter D(4) receptor-stimulated ERK. However, PP2 and the mitogen activated protein kinase kinase inhibitor PD98059 did not change D(4) receptor-mediated Akt/NF-kappa B activation. All these indicate that distinct mechanisms mediate ERK and Akt/NF-kappa B activation by D(4) dopamine receptor stimulation. We also demonstrated that D(4) receptor-stimulated cell proliferation is mediated by the Src/SHC/Ras/ERK pathway.     

A mitogen-activated protein kinase is involved in the inotropic but not chronotropic actions of adrenoceptor agonists and endothelin-1

The activation of mitogen-activated protein kinase (MAPK) pathways in the heart, for instance by alpha(1)-adrenoceptor agonists and endothelin-1, has primarily been associated with cellular growth regulation. Here we have investigated a possible role of MAPK pathways in the inotropic and chronotropic effects of adrenoceptor agonists and endothelin-1 in isolated rat left and right atria. Inotropic and chronotropic responses of the isolated atria to methoxamine, isoprenaline and endothelin-1 were measured in the absence and presence of inhibitors of MAPK pathways. The MAPK kinase (MKK(mek)) inhibitors PD98059 (100 microM) and U0126 (10 microM) significantly inhibited the inotropic responses to the alpha(1)-adrenoceptor agonist methoxamine (300 microM) and endothelin-1 (50 nM), but not the chronotropic responses to these agonists. U0126 but not PD98059 inhibited the inotropic response to 3 microM isoprenaline. None of the aforementioned inotropic and chronotropic effects were inhibited by the MAPKP(p38) inhibitor SB203580 (2 microM). We conclude that activation of the PD98059/U0126-sensitive MAPK pathway is essential for the inotropic but not chronotropic actions of adrenoceptor agonists and endothelin-1.     

Fundamental role of nitric oxide in neuritogenesis of PC12h cells

1 We investigated the neuritogenic action of nitric oxide (NO)-generating agents and their mechanisms of action in a subclone of rat pheochromocytoma, PC12h cells. 2 NO donors such as sodium nitroprusside (SNP, 0.05-1 microM), NOR1 (5-100 microM), NOR2 (5-20 microM), NOR3 (5-20 microM), NOR4 (5-100 microM), or S-nitroso-N-acetyl-DL-penicillamine (SNAP, 10-100 microM) significantly induced neurite outgrowth. 3 NOR4-induced neurite outgrowth was accompanied by expression of neurofilament 200 kDa subunit (NF200) protein, an axonal marker, and was significantly inhibited by an NO scavenger, a soluble GC inhibitor, and a PKG inhibitor: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (carboxy-PTIO, 20-100 microM), 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, 100 microM) and KT5823 (0.2-1 microM), respectively. 4 The intracellular cGMP concentration of cells was markedly increased by treatment with NOR4 (100 microM). 5 A mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059 (10-50 microM), abolished the NOR4-induced neurite outgrowth. In agreement with this observation, NOR4 did phosphorylate extracellular signal-regulated kinase (ERK) 1 and 2, substrates of MAPK kinase. 6 A membrane-permeable cGMP analog, 8-Br-cGMP (1 mM) also induced significant neurite outgrowth. The 8-Br-cGMP-induced neurite outgrowth was almost completely inhibited by both KT5823 (0.5 microM) and PD98059 (50 microM). Moreover, sustained ERK phosphorylation was observed in the 8-Br-cGMP-treated PC12h cells. 7 These results suggest that NO itself has the ability to induce neurite outgrowth and that NO-induced ERK activation involves the NO-cGMP-PKG signaling pathway in PC12h cells.     

ERK1/2信号通路在角质细胞生长因子-2诱导角膜基质细胞增殖中的作用

目的观察角质细胞生长因子(keratinocyte growthfactor-2,KGF-2)对角膜基质细胞的增殖作用及ERK1/2信号在角膜基质细胞增殖中的调控作用,探讨KGF-2对角膜基质细胞增殖的可能机制。方法体外培养角膜基质细胞,MTT法检测细胞增殖,Western blot检测磷酸化ERK1/2表达水平。结果 KGF-2在1～100 mg·L-1对角膜基质细胞有明显的促进增殖作用且呈现剂量-效应关系;100 mg·L-1 KGF-2处理角膜基质细胞,5、15、30 min后ERK1/2磷酸化表达水平明显升高,60、90、120、180 min后ERK1/2磷酸化表达水平逐渐减弱;20μmol.L-1 ERK1/2抑制剂PD98059可抑制KGF-2对角膜基质细胞的促增殖作用。结论 KGF-2激活ERK1/2信号通路,提高细胞增殖率,可被抑制剂PD98059阻断;ERK1/2信号通路调控角膜基质细胞的增殖,在角膜基质细胞损伤中发挥作用。     

p38 Mitogen-activated protein kinase regulates vasoconstriction in spontaneously hypertensive rats

We investigated whether p42/p44 mitogen-activated protein kinase (MAPK) and/or p38 MAPK participates in the regulation of vascular smooth muscle contraction by endothelin-1 (ET-1) in Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). ET-1 (10 nM) induced a sustained contraction in WKY and SHR aortas. PD98059 (100 microM), an inhibitor of p42/p44 MAPK kinase, partially attenuated the ET-1-induced contraction in WKY and SHR. However, SB203580 (10 microM), an inhibitor of p38 MAPK, relaxed the ET-1-induced contraction to the resting levels in SHR, but not in WKY. ET-1 (10 nM) increased phosphorylation of both p42/p44 MAPK and p38 MAPK in WKY and SHR. However, in SHR, p38 MAPK phosphorylation in response to ET-1 stimulation was increased more than in WKY. PD98059 (100 microM) and SB203580 (10 microM) abolished the phosphorylation of p42/p44 MAPK and p38 MAPK in response to ET-1 stimulation in WKY and SHR, respectively. On the other hand, SB203580 (10 microM) did not affect myosin light chain (MLC) phosphorylation in response to ET-1 (10 nM) stimulation in WKY and SHR. From these results, it is concluded that p42/p44 MAPK and/or p38 MAPK partially regulates the ET-1-induced vasoconstriction in WKY. However, p38 MAPK, rather than p42/p44 MAPK, activation plays an important role for the maintenance of ET-1-induced vasoconstriction in SHR through a MLC phosphorylation-independent pathway.     

PD98059对HL60细胞Ras-MEK1/2-ERK1/2信号转导影响的研究

目的探讨MEK1抑制剂PD98059对HL60细胞Ras-MEK1/2-ERK1/2信号转导以及细胞增殖的影响。方法四唑盐比色试验(MTT)法检测PD98059对HL60细胞增殖的抑制作用,流式细胞术观察PD98059对HL60细胞凋亡和G0/G1期阻滞的影响,半定量反转录-聚合酶链反应(RT-PCR)测定PD98059对HL60细胞ERK2、p27、Skp2基因表达的影响,免疫组织化学SP法检测ERK2、p27、Skp2蛋白表达的改变情况。结果 PD98059能够抑制HL60细胞的生长,该抑制作用具有浓度及时间依赖性(P<0.05)。PD98059能够促进HL60细胞凋亡,该作用具有浓度及时间依赖性(P<0.05);PD98059作用HL60细胞48h,随着浓度的增加G0/G1期阻滞增强(P<0.05)。PD98059可使HL60细胞ERK2、Skp2的mRNA及蛋白表达量减少,p27的mR-NA及蛋白表达量增加(P<0.05)。结论 PD98059能够抑制HL60细胞的生长,诱导细胞发生G0/G1期阻滞,促进其凋亡,这可能是通过PD98059阻断Ras-MEK1/2-ERK1/2信号转导,影响了ERK2、Skp2、p27等的表达而实现的。     

Contribution of the p38MAPK signalling pathway to proliferation in human cultured airway smooth muscle cells is mitogen-specific

We have investigated the role of p38MAPK in human airway smooth muscle (HASM) proliferation in response to thrombin and bFGF. The regulation of cyclin D1 mRNA, cyclin D1, cyclin E and p21Cip1 protein levels, and the extent of retinoblastoma protein (pRb) phosphorylation in response to activation of p38MAPK have also been examined. Two distinct inhibitors of p38MAPK, SB 203580 (10 microm) and SB 202190 (10 microm), prevented bFGF (0.3-3 nm)-stimulated cell proliferation, but had no effect on the response to thrombin (0.3-3 U ml(-1)). In cells incubated with thrombin or bFGF for 20 h, there was an increase in p38MAPK phosphorylation in response to bFGF, but not to thrombin. Thrombin and bFGF-stimulated increases in ERK phosphorylation and cyclin D1 mRNA and protein levels were not influenced by SB 203580 pre-treatment. Similarly, cyclin E and p21Cip1 protein levels, measured after 20 h incubation with mitogen, did not appear to be regulated by SB 203580 (10 microm). Although both thrombin and bFGF significantly increased levels of pRb phosphorylation, SB 203580 (10 microm) inhibited only bFGF-stimulated pRb phosphorylation. In addition, SB 203580 (10 microm) selectively inhibited bFGF-stimulated DNA synthesis, suggesting that the antimitogenic actions of SB 203580 on pRb phosphorylation cause cell cycle arrest at late G1 phase. In conclusion, these results indicate that p38MAPK is involved in bFGF-, but not in thrombin-stimulated HASM proliferation. The activation of the p38MAPK pathway by bFGF, but not by thrombin, regulates the phosphorylation of pRb without influencing cyclin D1 expression.     

Functional coupling of angiotensin II type 1 receptor with insulin resistance of energy substrate uptakes in immortalized cardiomyocytes (HL-1 cells)

BACKGROUND AND PURPOSE: Increased angiotensin II levels and insulin resistance coexist at the early stages of cardiomyopathies. To determine whether angiotensin II increases insulin resistance in cardiomyocytes, we studied the effect of angiotensin II on basal and insulin-stimulated transport rate of energy substrates in immortalized cardiomyocytes (HL-1 cells). EXPERIMENTAL APPROACH: Glucose and palmitic acid uptakes were measured using [(3)H]2-deoxy-D-glucose and [(14)C]palmitic acid, respectively, in cells exposed or not exposed to angiotensin II (100 nM), angiotensin II plus irbesartan or PD123319, type 1 and 2 receptor antagonists, or PD98059, an inhibitor of ERK1/2 activation. Cell viability, DNA, protein synthesis and surface area were evaluated by the MTT test, [(3)H]thymydine, [(3)H]leucine and morphometric analysis, respectively. Type 1 receptor levels were measured by western blot analysis. KEY RESULTS: Basal uptakes of glucose and palmitic acid by HL-1 cells (0.37+/-0.07 and 7.31+/-0.22 pmol per 10(4)cells per min, respectively) were both stimulated by 100 nM insulin (+91 and +64%, respectively). Cells exposed to angiotensin II remained viable and did not show signs of hypertrophy. In these conditions, the basal palmitic acid uptake of the cells increased (11.41+/-0.46 pmol per 10(4) cells per min) and insulin failed to stimulate the uptake of glucose and fatty acids. Changes in the rate of uptake of energy substrates were prevented or significantly reduced by irbesartan or PD98059. CONCLUSIONS AND IMPLICATIONS: Angiotensin II is a candidate for increasing insulin resistance in cardiomyocytes. Our results suggest a further mechanism for the cardiovascular protection offered by the angiotensin II type 1 receptor blockers.     

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+ -induced neuronal death in cultured hippocampal neurons

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb(2+) causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb(2+). Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb(2+)-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb(2+) treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 microM) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb(2+). And that Pb(2+)-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb(2+) and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.     

Phorbol 12-myristate 13-acetate triggers the protein kinase A-mediated phosphorylation and activation of the PDE4D5 cAMP phosphodiesterase in human aortic smooth muscle cells through a route involving extracellular signal regulated kinase (ERK)

Phosphodiesterase 4D5 is the sole PDE4D cAMP phosphodiesterase isoform expressed in human aortic smooth muscle cells (HASMC). Phorbol 12-myristate 13-acetate (PMA) challenge of HASMC rapidly activated PDE4D5 through a process ablated by the mitogen-activated protein kinase kinase inhibitor PD98059. PMA elicited an inhibitory effect on PDE4D5 activity in HASMC treated with the cyclooxygenase (COX) inhibitor indomethacin, the COX-2 selective inhibitor NS-398, the phospholipase A(2) inhibitor quinacrine, and the cAMP-dependent protein kinase A (PKA) inhibitor H89. PMA challenge of COS-1 cells elicited the rapid inhibition and phosphorylation of both recombinant and endogenous PDE4D5 in a manner ablated by PD98059 and not seen in S651A mutant PDE4D5. PMA promoted the generation of PGE(2) in the medium of HASMC and caused activation of both extracellular signal-regulated kinase (ERK) and PKA through a process ablated by indomethacin, NS-398, quinacrine, and PD98059. Exogenous prostaglandin (PG) E(2) increased cAMP levels and activated PKA in HASMC. COX-2 was expressed in HASMC but not in COS-1 cells. Forskolin challenge of COS-1 cells activated PDE4D5 by causing the PKA-mediated phosphorylation of Ser126 as detected using a novel phosphospecific antiserum. PMA challenge of HASMC elicited phosphorylation of the stimulatory PKA-specific phosphorylation site, Ser126 in PDE4D5 in a manner ablated by PD98059, indomethacin, and H89. We propose that, in HASMC, PMA activates PDE4D5 through an ERK-controlled autocrine mechanism. This involves PGE(2) generation, which causes activation of adenylyl cyclase, allowing PKA to elicit net activation of PDE4D5 by phosphorylation at Ser126.