A role for src tyrosine kinase in regulating adrenal aldosterone production

Adrenal aldosterone synthesis is influenced by a variety of factors. The major physiological regulators of aldosterone production are angiotensin II (Ang IotaIota) and potassium (K(+)). Ang IotaIota stimulates aldosterone production through the activation of multiple intracellular signaling pathways. It has recently been demonstrated that Ang IotaIota activates src tyrosine kinases in vascular smooth muscle cells. The src family of tyrosine kinases are widely distributed non-receptor kinases that influence several signal transduction pathways. In the present study we evaluated the effect of a selective src family inhibitor, PP2, on aldosterone production using a human adrenocortical carcinoma-derived (H295R) cell line. Treatments for 6 or 48 h with PP2 (0.3 microM-10 microM) inhibited basal, Ang IotaIota, K(+) and dibutyryladenosine cyclic monophosphate (dbcAMP) stimulation of aldosterone production in a concentration-dependent manner. PP2 did not affect cell viability at any of the concentrations tested. Moreover, time course studies using PP2 (10 microM) for 6, 12, 24, and 48 h revealed a time-dependent inhibition of aldosterone production. Inhibition by PP2 (0.3-10 microM) was also observed for the metabolism of 22R-hydroxycholesterol (22R-OHChol) to aldosterone in H295R cells. Since 22R-OHChol is a substrate for cytochrome P450 side-chain cleavage enzyme (CYP11A) that does not require steroidogenic acute regulatory (StAR) protein for transport to the inner mitochondrial membrane, these results suggest that PP2 inhibition occurred beyond the rate-limiting step in aldosterone synthesis. Genistein, a non-specific tyrosine kinase inhibitor also blocked aldosterone production, but the inhibition was the result of a non-specific effect on 3beta-hydroxysteroid dehydrogenase (3betaHSD). In contrast, PP2 did not appear to act as a direct inhibitor of 3betaHSD activity. To further investigate the site of PP2 action, we examined its effect on H295R cell metabolism of [(14)C]progesterone using thin layer chromatography. PP2 treatment for 48 h caused an increase in the conversion of progesterone to 17alpha-hydroxyprogesterone. To determine if this apparent increase in 17alpha-hydroxylase activity was due to increased transcript, we examined the effect of PP2 on CYP17 mRNA. PP2 treatment caused an increase in CYP17 mRNA without an effect on 3betaHSD mRNA levels. Inhibition of protein synthesis with cycloheximide increased basal levels of CYP17 mRNA levels and blocked the induction observed by PP2. This suggests that new protein synthesis is a necessary part of PP2 induction of CYP17. Taken together these data suggest that the src tyrosine kinase inhibitor, PP2, is a potent inhibitor of aldosterone production. One mechanism for the inhibition is through an induction of CYP17 mRNA and enzyme activity. Src tyrosine kinases, therefore, may be involved with the promotion of a glomerulosa phenotype through the inhibition of CYP17 expression.

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ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00474.2002 ◽

2002 ◽

Vol 283 (6) ◽

pp. H2322-H2330 ◽

Cited By ~ 74

Author(s):

Thomas Krieg ◽

Qining Qin ◽

Elizabeth C. McIntosh ◽

Michael V. Cohen ◽

James M. Downey

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Pi3 Kinase ◽

Dependent Manner ◽

Src Tyrosine Kinase ◽

Akt Phosphorylation ◽

Downstream Target

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 ± 0.33 fold ( P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 ± 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, Gi protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.

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Temporal regulation of the IgE-dependent 1,2-diacylglycerol production by tyrosine kinase activation in a rat (RBL 2H3) mast-cell line

Biochemical Journal ◽

10.1042/bj2990109 ◽

1994 ◽

Vol 299 (1) ◽

pp. 109-114 ◽

Cited By ~ 22

Author(s):

P Lin ◽

S J Fung ◽

S Li ◽

T Chen ◽

B Repetto ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Kinases ◽

Kinase Inhibitor ◽

Initial Increase ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Kinase Activation ◽

Temporal Regulation ◽

Mast Cell Line

We explored the possible role of tyrosine kinases in the IgE-dependent regulation of 1,2-diacylglycerol (DAG) production in RBL 2H3 cells. When triggered via their high-affinity IgE receptors (Fc epsilon RI), there was a rapid phosphorylation of tyrosine residues on a number of proteins. The phosphorylation of these proteins and ultimately histamine release were inhibited in a concentration-dependent manner by the tyrosine kinase inhibitor, tyrphostin. In cells labelled with [3H]myristic acid, we observed a characteristic biphasic increase in [3H]DAG production. In the presence of tyrosine kinase inhibitor, the initial increase in DAG was still observed, but the secondary increase, which was dependent on phosphatidylcholine-specific phospholipase D (PC-PLD) activation, was completely abolished. Tyrphostin significantly inhibited IgE-dependent activation of PC-PLD, suggesting that PC-PLD activation was regulated by tyrosine phosphorylation. Furthermore, when proteins from RBL 2H3 cells were immunoprecipitated with an anti-phosphotyrosine antibody, PC-PLD activity was recovered from the immunoprecipitated fraction. These results demonstrate that the secondary, but not the initial, phase of 1,2-DAG production in response to Fc epsilon RI aggregation is regulated by the initial activation of tyrosine kinases and that PC-PLD may be regulated directly by this mechanism.

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Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00429.2001 ◽

2002 ◽

Vol 283 (4) ◽

pp. G893-G899 ◽

Cited By ~ 5

Author(s):

Monica C. Chen ◽

Travis E. Solomon ◽

Eduardo Perez Salazar ◽

Robert Kui ◽

Enrique Rozengurt ◽

...

Keyword(s):

Gastric Mucosa ◽

Epithelial Cells ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Phosphorylation ◽

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Paracellular Permeability ◽

Maximal Response ◽

Src Tyrosine Kinase

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416 was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 μM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416 in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.

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TEL/PDGFβR Induces Hematologic Malignancies in Mice That Respond to a Specific Tyrosine Kinase Inhibitor

Blood ◽

10.1182/blood.v93.5.1707 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1707-1714 ◽

Cited By ~ 80

Author(s):

Michael H. Tomasson ◽

Ifor R. Williams ◽

Robert Hasserjian ◽

Chirayu Udomsakdi ◽

Shannon M. McGrath ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Transgenic Animals ◽

Myeloid Lineage ◽

Protein Tyrosine

Abstract The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.

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603 POSTER Effect of the SRC tyrosine kinase inhibitor dasatinib in combination with erlotinib and in cells with acquired resistance to erlotinib

European Journal of Cancer Supplements ◽

10.1016/s1359-6349(06)70608-5 ◽

2006 ◽

Vol 4 (12) ◽

pp. 182

Author(s):

E. Haura ◽

J. Gao ◽

J. Li

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Kinase Inhibitor ◽

Acquired Resistance ◽

Src Tyrosine Kinase ◽

In Cells

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Src Tyrosine Kinase Inhibitor, M475271, Suppresses Subcutaneous Growth and Production of Lung Metastasis Via Inhibition of Proliferation, Invasion, and Vascularization of Human Lung Adenocarcinoma Cells

Clinical & Experimental Metastasis ◽

10.1007/s10585-005-7768-5 ◽

2005 ◽

Vol 22 (3) ◽

pp. 195-204 ◽

Cited By ~ 11

Author(s):

Rui Zheng ◽

Seiji Yano ◽

Yuka Matsumori ◽

Emiko Nakataki ◽

Hiroaki Muguruma ◽

...

Keyword(s):

Tyrosine Kinase ◽

Lung Adenocarcinoma ◽

Tyrosine Kinase Inhibitor ◽

Human Lung ◽

Kinase Inhibitor ◽

Src Tyrosine Kinase ◽

Inhibition Of Proliferation ◽

Adenocarcinoma Cells ◽

Human Lung Adenocarcinoma Cells ◽

Lung Adenocarcinoma Cells

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Development of a novel bone-targeted Src tyrosine kinase inhibitor AP23451 having potent in vitro and in vivo anti-tesorptive properties

European Journal Of Haematology ◽

10.1034/j.1600-0609.2003.11718.x ◽

2003 ◽

Vol 70 (4) ◽

pp. 271-271

Author(s):

W. Shakespeare

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Kinase Inhibitor ◽

Src Tyrosine Kinase

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The Tyrosine Kinase Inhibitor Adaphostin (NSC 680410), but Not Imatinib Mesylate, Inhibits Survival and Src Tyrosine Kinase Family- Triggered Signaling Pathways of MM Cells.

Blood ◽

10.1182/blood.v104.11.3352.3352 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3352-3352

Author(s):

Klaus Podar ◽

Melissa Simoncini ◽

Yu-Tzu Tai ◽

Martin Sattler ◽

Kenji Ishitsuka ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Signaling Pathways ◽

Imatinib Mesylate ◽

Kinase Inhibitor ◽

K562 Cells ◽

Parp Cleavage ◽

Src Tyrosine Kinase ◽

Kinase Family ◽

Long Term Treatment

Abstract The tyrosine kinase inhibitor adaphostin is a member of the tyrophostin family of small molecules that interfere with peptide binding rather, than targeting the kinase ATP-binding site. Adaphostin has therefore been examined as an alternative to the 2-phenylaminopyrimidine derivate imatinib mesylate, with remarkable efficacy in the treatment of chronic myeloic leukemia (CML). Previous studies show that adaphostin induces apoptosis: (1) in Bcr/Abl+ cells more rapidly than imatinib mesylate; (2) in imatinib mesylate resistant cells; and (3) in Bcr/ Abl - cells. Imatinib mesylate has minimal, if any activity in MM; the efficacy of adaphostin in multiple myeloma (MM) is unknown. Here we compare the effects of adaphostin and imatinib mesylate against human MM cells. Our results show concentration-dependent apoptosis in MM.1S, U266, OPM-2, INA-6, RPMI8226 and RPMI-Dox40 MM cells after treatment with adaphostin, but not with imatinib mesylate. Imatinib mesylate induced more than 50% apoptosis in K562 cells using concentrations as low as 1mM, which served as a positive control. Moreover, adaphostin, but not imatinib mesylate, induced caspase-9, caspase-8, and PARP cleavage, as well as downregulation of Mcl-1, in MM cells. Further results demonstrated that adaphostin induces peroxide production and DNA strand breaks after long-term treatment. Importantly MM cell proliferation induced by MM cell binding to BMSCs was abrogated by adaphostin- treatment. IL-6 and IGF-1 signaling and sequelae triggered by these cytokines are important growth, survival, and drug resistance factors in MM; conversely, adaphostin but not imatinib mesylate, inhibited phosphorylation of Src tyrosine kinase family, Akt-1, and ERK. Taken together, our studies in MM cells show that (1) adaphostin- inhibits IGF-1- and IL-6- triggered signaling pathways as well as (2) induces reactive oxygen species and apoptosis. These studies therefore provide the preclinical framework for its clinical evaluation to improve patient outcome in MM.

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