Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI<sub>2</sub> release but has no effect on von Willebrand factor secretion or E‐selectin expression

Wheeler‐Jones, Caroline P.D.; May, Michael J.; Houliston, Rebecca A.; Pearson, Jeremy D.

doi:10.1016/0014-5793(96)00547-9

Cited by 43 publications

(36 citation statements)

References 27 publications

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“…In platelets, this effect does not alter arachidonic acid release nor affect aggregation (45). In contrast, PD098059 blocks prostacyclin I 2 release in thrombinstimulated endothelial cells while not affecting van Willebrand factor secretion (46). Thus, blockade of thrombin signaling events in different cell types leads to dramatically different results upon arachidonic acid metabolism.…”

Section: Discussionmentioning

confidence: 98%

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

Favata¹,

Horiuchi²,

Manos³

et al. 1998

Journal of Biological Chemistry

2,844

2,077

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We further demonstrate that the two compounds bind to ⌬N3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for ⌬N3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the ⌬N3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

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Section: Discussionmentioning

confidence: 98%

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

Favata¹,

Horiuchi²,

Manos³

et al. 1998

Journal of Biological Chemistry

2,844

2,077

View full text Add to dashboard Cite

show abstract

“…Proteins of interest within whole granulosa-cell lysates were separated by 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) (Wheeler-Jones et al 1996). After electrophoresis, proteins were transferred to an Immobilon-polyvinylidene difluoride (PVDF) membrane, using the Bio-Rad semidry blotting system.…”

Section: Sds-page and Western Immunoblottingmentioning

confidence: 99%

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

Chin¹,

Abayasekara²

2004

Journal of Endocrinology

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The corpus luteum formed after luteinization of follicular cells secretes progesterone under the control of luteinizing hormone (LH). Binding of LH to its G-protein-coupled receptor leads to the activation of the adenylate cyclase/ cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) signalling pathway. The identification of a new class of cAMP-binding proteins termed 'guanine nucleotide exchange factors' (cAMP-GEFs) provides a means by which changes in cAMP could yield actions that are independent of PKA. Hence, in this study, we have explored the hypothesis that steroidogenesis in luteinizing cells is mediated in both a cAMP/PKA-dependent and cAMP-dependent, but PKA-independent, manner. Human granulosa cells were isolated from follicular aspirates of women undergoing assisted conception. Luteinizing human granulosa cells were cultured for up to 3 days in the presence of human (h)LH and the adenylate cyclase activator forskolin in the added presence or absence of increasing doses of the PKA inhibitors H89 (N-[2-(4-bromocinnamylamino)ethyl] 5-isoquinoline) and PKI (myristoylated protein kinase A inhibitor amide 14-22) or the cAMP antagonist, Rp-cAMP. Agonist-stimulated progesterone secretion was inhibited in a dose-dependent manner by the PKA inhibitors and the cAMP antagonist, with decreasing sensitivity as luteinization progressed. Pretreatment of granulosa cells for 4 h with human (h)LHreduced the effectiveness of H89 in inhibiting progesterone secretion. Under basal conditions, cAMP-GEFI expression increased progressively throughout culture, and this could be further enhanced when cells were incubated with increasing doses of LH and forskolin. Furthermore, incubation of cells in the presence of increasing concentrations of the novel cAMP-GEF-specific cAMP analogue, 8 CPT-2 ME-cAMP (8-(4-chloro-phenylthio)-2 -0-methyladenosine-3 ,5 -cyclic monophosphate), increased progesterone secretion in a dose-dependent manner. The results show that increases in cAMP generated by LH and forskolin, in addition to activating PKA, also induce increases in cAMP-GEFI protein expression in luteinizing human granulosa cells. In addition, activation of cAMP-GEFI results in increased progesterone secretion. Hence, increases in cAMP lead to the activation of PKA-dependent, as well as PKA-independent but cAMPdependent (via cAMP-GEFI), signalling mechanisms. Since cAMP-GEFs have the capacity to activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signalling pathways, these may provide the potential mechanisms by which cAMP-dependent but PKAindependent progesterone synthesis is regulated.

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“…Whole granulosa-cell lysates proteins were separated by 10% SDS-PAGE (Wheeler-Jones et al 1996). After electrophoresis, proteins were transferred to Immobilonpolyvinylidene difluoride (PVDF) membrane by the BioRad semidry blotting system.…”

Section: Sds-page and Western Immunoblottingmentioning

confidence: 99%

Changes in cAMP-dependent protein kinase (PKA) and progesterone secretion in luteinizing human granulosa cells

Chin

Harris

Abayasekara³

2004

Journal of Endocrinology

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Luteinization of follicular granulosa cells leads to an increase in progesterone secretion that is regulated by luteinizing hormone (LH). LH acts mainly by elevating intracellular cyclic 3 ,5 -adenosine monophosphate (cAMP) and activating cAMP-dependent protein kinase (PKA). In this study, we have examined the role of PKA in relation to progesterone output by luteinizing human granulosa cells. Human granulosa cells were obtained by percoll gradient centrifugation of follicular aspirates of patients undergoing oocyte retrieval for assisted conception. Cells were cultured in serum-supplemented medium for up to 3 days in the presence and/or absence of human (h)LH and other cAMP-elevating agents. Spent medium was assayed for cAMP and progesterone content by specific RIA. Cell lysates were collected and assessed for PKA regulatory (R)II /catalytic (C) expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RII /C expression appeared to increase throughout the 3-day culture period. In the presence of hLH and other cAMP-elevating agents, progesterone secretion increased in a dose-dependent manner coincident with an increase in cAMP. However, despite the increase in both progesterone secretion and cAMP accumulation, there was a dose-dependent decrease in both PKA RII and C expression. Thus, data presented in this study show that increases in progesterone secretion in luteinizing human granulosa cells can be dissociated from increases in PKA expression. This notion implies that progesterone secretion may be regulated by PKA-dependent as well as PKAindependent mechanisms.

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Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI₂ release but has no effect on von Willebrand factor secretion or E‐selectin expression

Cited by 43 publications

References 27 publications

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

Changes in cAMP-dependent protein kinase (PKA) and progesterone secretion in luteinizing human granulosa cells

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Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E‐selectin expression

Cited by 43 publications

References 27 publications

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

Changes in cAMP-dependent protein kinase (PKA) and progesterone secretion in luteinizing human granulosa cells

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Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI₂ release but has no effect on von Willebrand factor secretion or E‐selectin expression