p38MAPK inhibition enhances basal and norepinephrine-stimulated p42/44MAPK phosphorylation in rat pinealocytes

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4beta phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.     

Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland

Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.     

Proinflammatory cytokines,IL-1β and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts

The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1β (17-fold) and TNF-α (74-fold). IL-1β and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1β- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1β- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1β and TNF-α by up to 7- and 11-fold, respectively. IL-1β- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1β and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.     

Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.     

Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone

The aim of these studies was to identify the signaling mechanism(s) that contribute to GnRH-induced expression of MAPK phosphatase (MKP)-2, a dual specificity phosphatase that selectively inactivates MAPKs. GnRH receptor activation induced MKP-2 expression in both clonal (alphaT3-1) and primary gonadotropes. Activation of PKC isozymes was sufficient and required for MKP-2 induction. Inhibition of the extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) but not the p38 MAPK cascade was sufficient to block GnRH-induced MKP-2 expression. Induction of MKP-2 by GnRH was dependent on elevation in intracellular Ca(2+). Inhibition of Ca(2+) influx through L-type voltage-gated calcium channels blocked GnRH-induced MKP-2 expression. Depletion of intracellular Ca(2+) stores with thapsigargin blocked MKP-2 activation by GnRH independent of ERK and JNK activity. These results support the conclusion that MKP-2 induction by GnRH occurs via MAPK-dependent and -independent pathways. One mechanism requires GnRH-induced ERK and JNK activation, while a second MAPK-independent pathway requires a thapsigargin-sensitive calcium signal.     

Apoptosis induction by p38 MAPK inhibitor in human colon cancer cells

BACKGROUND/AIMS: The p38 mitogen-activated protein kinases (p38 MAPKs) function in a wide variety of signaling pathways. However, the role of p38s is cell type- and stimulus-dependent. The present study aimed to evaluate the effects of p38 MAPK inhibitor on human colon cancer cells. METHODOLOGY: The effect of p38 MAPK inhibitor, FR167653, on DLD-1 and SW480 was investigated related to cell proliferation, apoptosis induction and caspase activity. Additionally, the effect of FR167653 on colon cancer cell migration, MMPs production and ability to adhere to extracellular matrix was investigated. RESULTS: Inhibitor of p38 MAPK dose-dependently suppressed the proliferative activity of both cell lines, and increased the induction of cell apoptosis. The caspase-3, 8, and 9 activities were accompanied in the pathway. Neither cell migration, MMPs production, nor the ability to adhere extracellular matrix were affected by FR167653. CONCLUSIONS: Inhibitor of p38 MAPK suppressed the proliferation of colon cancer cells by induction of cell apoptosis through the caspase activation. The present results suggest the pro-oncogenic role ofp38 in colon cancer, and its inhibition would be a novel strategy for the prevention and treatment of colon cancer.     

Simvastatin potenciates PGI(2) release induced by HDL in human VSMC: effect on Cox-2 up-regulation and MAPK signalling pathways activated by HDL

High density lipoproteins (HDL) induce prostacyclin (PGI(2)) release in vascular smooth muscle cells (VSMC) by up-regulation of cyclooxygenase-2 (Cox-2). Our goal was to analyse the mechanisms underlying this effect, and its potential modulation by HMG-CoA reductase inhibition in human VSMC. The contribution of mitogen-activated protein kinase (MAPK) signalling pathways was assessed by Western blot analysis and using specific inhibitors [PD098059 for p42/44 MAPK kinase (MEK); SB203580 for p38 MAPK or L-JNKI1 for c-Jun N-terminal kinase-1 (JNK-1)]. HDL-induced PGI(2) release was inhibited by rofecoxib (a specific Cox-2 inhibitor, 5 microM). HDL induced the early activation of p42 MAPK, p38 MAPK and JNK-1. p42/44 MAPK was the major pathway involved in both Cox-2 up-regulation and PGI(2) synthesis; p38 MAPK was also involved in both processes while JNK inhibition only affected PGI(2) synthesis. Pertussis toxin (an inhibitor of Galphai/Galphao proteins) prevented MAPK activation and inhibited both Cox-2 up-regulation and PGI(2) release. Genistein (a tyrosine kinase inhibitor) inhibited PGI(2) release without affecting MAPK activation or Cox-2 up-regulation. Simvastatin (0.1-1 microM) increased HDL-induced PGI(2) release ( approximately 45% at 1 microM) but did not significantly modify early MAPK activation or Cox-2 expression. Simvastatin alone did not significantly affect PGI(2) release. Our results suggest that mechanisms associated with G protein-coupled receptor activation, trigger Cox-2 up-regulation and PGI(2) release via multiple MAPK signalling pathways in VSMC. The mechanism is independent of tyrosine kinase receptors, although cytosolic tyrosine kinases could activate Cox-2 post-translationally. The potential contribution of HDL to vascular homeostasis, via increases in PGI(2) synthesis, could be enhanced by HMG-CoA reductase inhibitors.     

Adrenocorticotropin hormone (ACTH) effects on MAPK phosphorylation in human fasciculata cells and in embryonic kidney 293 cells expressing human melanocortin 2 receptor (MC2R) and MC2R accessory protein (MRAP)β

Adrenocorticotropin hormone (ACTH) exerts trophic effects on adrenocortical cells. We studied the phosphorylation of mitogen-activated proteins kinases (MAPKs) in human embryonic kidney cells stably expressing the ACTH receptor, MC2R, and its accessory protein MRAPβ and in primary cultures of human adrenal fasciculata cells. ACTH induced a maximal increase in p44/p42(mapk) and of p38 MAPK phosphorylation after 5min. Neither the overexpression of wild-type arrestin2, arrestin3 or their respective dominant negative forms affected p44/p42(mapk) phosphorylation. However, preincubation with the recycling inhibitors brefeldin A and monensin attenuated both cAMP accumulation and p44/p42(mapk) phosphorylation proportionally. Cyclic AMP-related PKA inhibitors (H89, KI(6-22)) and Rp-cAMPS decreased p44/p42(mapk) phosphorylation but not ACTH-mediated cAMP production. The selective Epac1/2 activator, 8-pCPT-2'-O-MecAMP, did not modify the effect of ACTH. Thus, cAMP/PKA, but not cAMP/Epac1/2 pathways, or arrestin-coupled internalization of MC2R is involved in ACTH-induced p44/p42(mapk) phosphorylation by human MC2R. Together, ACTH binding to MC2R stimulates PKA-dependent p44/p42(mapk) phosphorylation.     

Inhibition of stress-activated p38 mitogen-activated protein kinase induces low-density lipoprotein receptor expression

We have recently shown that different signal transduction pathways initiated by a variety of agents converge on growth-responsive p42/44MAPK signaling cascade to induce low-density lipoprotein (LDL) receptor expression. Our recent demonstration that stress-activated p38MAPK negatively regulates LDL receptor expression in an isoform-specific manner via modulation of p42/44MAPK cascade represents a new dimension of complexity in the molecular communication that governs LDL receptor expression. The suggested one-way communication between p38MAPK and p42/44MAPK provides a potential mechanism for fine-tuning cellular levels of cholesterol in response to a diverse set of environmental cues, including stress. Cross talk between MAPKs opens new avenues toward understanding a variety of pathogenic processes; this makes them tempting targets for therapeutic interventions in cardiovascular diseases.     

The phospholipids sphingosine-1-phosphate and lysophosphatidic acid prevent apoptosis in osteoblastic cells via a signaling pathway involving G(i) proteins and phosphatidylinositol-3 kinase

The naturally occurring phospholipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have recently emerged as bioactive compounds that exert mitogenic effects in many cell types, including osteoblasts. In the current study, we examined the ability of each of these compounds to influence osteoblast survival. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling and DNA fragmentation assays, we found that both LPA and S1P dose-dependently inhibited (by at least 50% and 40%, respectively) the apoptosis induced by serum withdrawal in cultures of primary calvarial rat osteoblasts and SaOS-2 cells. The antiapoptotic effects were inhibited by pertussis toxin, wortmannin, and LY294002, implicating G(i) proteins and phosphatidylinositol-3 kinase (PI-3 kinase) in the signaling pathway that mediates phospholipid-induced osteoblast survival. Specific inhibitors of p42/44 MAPK signaling did not block LPA- or S1P-induced osteoblast survival. LPA and S1P induced PI-3 kinase-dependent activation of p70 S6 kinase, but rapamycin, a specific inhibitor of p70 S6 kinase activation, did not prevent phospholipid-induced osteoblast survival. LPA and S1P also inhibited apoptosis in Swiss 3T3 fibroblastic cells in a G(i) protein-dependent fashion. In fibroblastic cells, however, the antiapoptotic effects of S1P were sensitive to inhibition of both PI-3 kinase and p42/44 MAPK signaling, whereas those of LPA were partially abrogated by inhibitors of p42/44 MAPK signaling but not by PI-3 kinase inhibitors. These data demonstrate that LPA and S1P potently promote osteoblast survival in vitro, and that cell-type specificity exists in the antiapoptotic signaling pathways activated by phospholipids.     

Ethanol-inhibited [3H]thymidine incorporation via protein kinase C-p44/42 mitogen-activated protein kinase/phospholipase A2 signal pathway in renal proximal tubule cells

BACKGROUND: Ethanol exposure leads to changes of cell proliferation in a variety of cell types. However, how ethanol affects the proliferation of renal proximal tubule cells is not known. METHODS: To examine the effect of ethanol on cell proliferation and its related signaling pathway, [H]thymidine incorporation, release of [H]arachidonic acid (AA), and Western blotting of protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) were performed in primary cultured rabbit renal proximal tubule cells. RESULTS: Ethanol inhibited [H]thymidine incorporation in a time- and dose-dependent manner. An inhibitory effect of ethanol on [H]thymidine incorporation was predominantly observed after 12 hr of treatment with 100 mM ethanol. Ethanol increased AA release and prostaglandin E2 production. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by phospholipase A2 inhibitors and was significantly blocked by PKC inhibitors. Indeed, ethanol induced a PKC translocation from the cytosolic to the membrane fraction. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by PD 98059 (a p44/42 MAPK inhibitor), but not by SB 203580 (a p38 MAPK inhibitor), and ethanol increased the phosphorylation of p44/42 MAPK. Results of phosphorylated p44/42 MAPK by ethanol were consistent with those of [H]thymidine incorporation and [H]AA-release experiments. CONCLUSIONS: Ethanol inhibited [H]thymidine incorporation via PKC, p44/42 MAPK, and phospholipase A2 signaling pathways in primary cultured renal proximal tubule cells.     

Mechanism of prostaglandin E2-stimulated heat shock protein 27 induction in osteoblast-like MC3T3-E1 cells

We investigated the effect of prostaglandin E2 (PGE2) on the induction of heat shock protein 27 (HSP27) and HSP70, and the mechanism behind the induction in osteoblast-like MC3T3-E1 cells. PGE2 time-dependently increased the level of HSP27 without affecting the level of HSP70. PGE2 stimulated the accumulation of HSP27 dose-dependently in the range between 10 nM and 10 microM. PGE2 stimulated the increase in the level of the mRNA for HSP27. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the PGE2-induced HSP27 accumulation. The effect of PGE2 on HSP27 accumulation was reduced in the PKC down-regulated cells. BAPTA/AM, a chelator of intracellular Ca2+, or TMB-8, an inhibitor of intracellular Ca2+ mobilization, reduced the accumulation of HSP27 induced by PGE2. Dibutyryl cAMP had little effect on the basal level of HSP27. PGE2 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, reduced the accumulation of HSP27 induced by PGE2. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the HSP27 accumulation induced by PGE2. U-73122, an inhibitor of phospholipase C, and calphostin C reduced the PGE2-induced phosphorylation of both p44/p42 MAP kinase and p38 MAP kinase. These results indicate that PGE2 stimulates the induction of HSP27 through PKC-dependent activations of both p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.     

Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.     

Anti‐leukaemic activity of the TYK2 selective inhibitor NDI‐031301 in T‐cell acute lymphoblastic leukaemia

Activation of tyrosine kinase 2 (TYK2) contributes to the aberrant survival of T‐cell acute lymphoblastic leukaemia (T‐ALL) cells. Here we demonstrate the anti‐leukaemic activity of a novel TYK2 inhibitor, NDI‐031301. NDI‐031301 is a potent and selective inhibitor of TYK2 that induced robust growth inhibition of human T‐ALL cell lines. NDI‐031301 treatment of human T‐ALL cell lines resulted in induction of apoptosis that was not observed with the JAK inhibitors tofacitinib and baricitinib. Further investigation revealed that NDI‐031301 treatment uniquely leads to activation of three mitogen‐activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage. Activation of p38 MAPK occurred within 1 h of NDI‐031301 treatment and was responsible for NDI‐031301‐induced T‐ALL cell death, as pharmacological inhibition of p38 MAPK partially rescued apoptosis induced by TYK2 inhibitor. Finally, daily oral administration of NDI‐031301 at 100 mg/kg bid to immunodeficient mice engrafted with KOPT‐K1 T‐ALL cells was well tolerated, and led to decreased tumour burden and a significant survival benefit. These results support selective inhibition of TYK2 as a promising potential therapeutic strategy for T‐ALL.     

GSTT1 is upregulated by oxidative stress through p38-MK2 signaling pathway in human granulosa cells: possible association with mitochondrial activity

We previously reported that GSTT1 was upregulated in human granulosa cells during aging and that activation and localization of p38 MAPK was changed in parallel. Although oxidative stress is responsible for these changes, the age-associated expression of GSTT1 regulated by MAPKs and the role of GSTT1 in aged granulosa cells remain unclear. Therefore, we examined the relationship between the expression of GSTT1 and MAPK signaling pathways using human granulosa-like KGN cells stimulated with H(2)O(2) in the presence or absence of various MAPK inhibitors. Interestingly, H(2)O(2)-induced GSTT1 was only inhibited by a p38 inhibitor. An inhibitor of MK2, a downstream regulator of p38, also diminished H(2)O(2)-induced GSTT1 upregulation. Notably, both p38 and MK2 were significantly inactivated in cells carrying an shRNA construct of GSTT1 (?GSTT1 cells), suggesting that the p38-MK2 pathway is essential for age-associated upregulation of GSTT1. The relevance of GSTT1 in mitochondrial activity was then determined. ?GSTT1 cells displayed enhanced polarization of mitochondrial membrane potential without increasing the apoptosis, suggesting that the age-associated upregulation of GSTT1 may influence the mitochondrial activity of granulosa cells.     

Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21(waf1/cip1) in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like alpha-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 microM, and DFMO elicited effects at concentrations of 100 microM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21(waf1/cip1). This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21(waf1/cip1) depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21(waf1/cip1) were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21(waf1/cip1) were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21(waf1/cip1) and consequent inhibition of cell proliferation.     

Downregulation of angiotensin-converting enzyme by tumor necrosis factor-alpha and interleukin-1beta in cultured human endothelial cells.

Angiotensin-converting enzyme (ACE) and cytokines are considered to play an important role in the pathophysiology of cardiovascular diseases such as atherosclerosis. In the present study, the effects of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on ACE in cultured human umbilical vein endothelial cells (HUVECs) was studied. TNF-alpha (0.1-10 ng/ml) and IL-1beta (0.1-10 ng/ml) caused a dose- and time-dependent decrease in the amount of ACE in intact endothelial cell membranes and decreased levels of ACE mRNA. TNF-alpha and IL-1beta activated p44/42 and p38 mitogen-activated protein kinases (MAPKs) in HUVECs; this was inhibited by the specific inhibitors of these kinases, PD98059 and SB202190, respectively. Pretreatment of endothelial cells with the specific p38 MAPK inhibitor SB202190 (5 microM) or hydrocortisone (5 microM) partly reversed the suppression of ACE by TNF-alpha or IL-1beta, whereas the specific p44/42 MAPK inhibitor PD98059 (40 microM) was without effect. Vascular endothelial growth factor (1 ng/ml) caused an increase in membrane-bound ACE and ACE mRNA levels which was inhibited by pretreatment of the cells with TNF-alpha (1 ng/ml) or IL-1beta (1 ng/ml). In summary, the cytokines TNF-alpha and IL-1beta downregulated ACE in cultured human endothelial cells, which effect was probably mediated by the p38 MAPK pathway. Downregulation of ACE by TNF-alpha and IL-1beta locally in the vascular wall may be a counterbalancing mechanism in inflammatory processes such as atherosclerosis, leading to decreased production of angiotensin II and accumulation of bradykinin.