scholarly journals Adenosine 5′-Monophosphate-Activated Protein Kinase Regulates Progesterone Secretion in Rat Granulosa Cells

Endocrinology ◽  
2005 ◽  
Vol 146 (10) ◽  
pp. 4500-4513 ◽  
Author(s):  
Lucie Tosca ◽  
Pascal Froment ◽  
Patricia Solnais ◽  
Pascal Ferré ◽  
Fabienne Foufelle ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Dominant Negative ◽  
Detectable Effect ◽  
Mrna Levels ◽  
Progesterone Secretion ◽  
Igf I ◽  
Amp Activated Protein Kinase ◽  
Acetyl Coenzyme A Carboxylase ◽  
Dose Dependent

The AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism involved in fatty acid and cholesterol synthesis. In the ovary, cholesterol plays a key role in steroid production. We report the presence of AMPK in rat ovaries, and we have investigated its role in granulosa cells. We show using RT-PCR and Western blot that the mRNAs for the α1/2 and β1/2 subunits and the proteins are found in the ovaries. Immunohistochemistry localized the α1 AMPK subunit in granulosa cells, corpus luteum, and oocyte and less abundantly in theca cells. Treatment with 1 mm 5-amino-imidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR), an activator of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKα1 on Thr172 in primary granulosa cells. Simultaneously, phosphorylation of acetyl-coenzyme A carboxylase at Ser79 was also increased. AICAR treatment for 48 h halved progesterone secretion, 3β-HSD protein and mRNA levels, and phosphorylation of both basal MAPK ERK1/2 and p38 and in response to IGF-I and/or FSH in granulosa cells. AICAR treatment (1 mm) had no detectable effect on basal and FSH- and/or IGF-I-induced estradiol production and on granulosa cell proliferation or viability. Adenovirus-mediated expression of dominant negative AMPK totally abolished the effects of AICAR on progesterone secretion, 3β-HSD protein production, and MAPK ERK1/2 and p38 phosphorylation. Moreover, we showed using specific in- hibitors of ERK1/2 and p38 MAPK that the MAPK ERK1/2 and not p38 is involved in progesterone secretion and 3β-HSD expression, strongly suggesting that the activation of AMPK in response to AICAR reduces progesterone production through the MAPK ERK1/2 signaling pathway in rat granulosa cells.

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

2004 ◽  
Vol 183 (1) ◽  
pp. 51-60 ◽  
Author(s):  
E C Chin ◽  
D R E Abayasekara
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Independent Manner ◽  
Human Granulosa Cells ◽  
Progesterone Secretion ◽  
Dose Dependent

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)LH reduced the effectiveness of H89 in inhibiting progester-one 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 cAMP-dependent (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 PKA-independent progesterone synthesis is regulated.

scholarly journals AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles

2006 ◽  
Vol 190 (1) ◽  
pp. 85-97 ◽  
Author(s):  
Lucie Tosca ◽  
Sabine Crochet ◽  
Pascal Ferré ◽  
Fabienne Foufelle ◽  
Sophie Tesseraud ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Cholesterol Metabolism ◽  
Specific Inhibitor ◽  
Negative Regulator ◽  
Star Protein ◽  
Theca Cells ◽  
Progesterone Secretion ◽  
Preovulatory Follicles ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a fuel sensor in glucose, lipid, and cholesterol metabolism. Using RT-PCR and Western blot, AMPK subunits mRNAs (α1/2, β1/2, and γ1/2) and proteins (α1/2 and β1/2) can be found in the hen preovulatory follicles and precisely in both granulosa and theca cells. These preovulatory follicles are organized in a hierarchy according to their size (F5/6 to F1). The smallest number (F1) corresponds to the largest size and the latest mature stage. Phosphorylation of AMPKα on Thr172 and of acetyl-CoA carboxylase on Ser79 are higher in F4 and F3 than in F1 granulosa cells. However, they are not affected in F4–F1 theca cells. Treatment with 1 mM 5-amino-imidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR), an activator of AMPK, dose dependently increased phosphorylation of AMPKα on Thr172 in primary F3/4 and F1 granulosa cells. In the absence of FSH, AICAR treatment increased progesterone, P450 side chain cleavage and steroidogenic acute regulatory (StAR) production in both F3/4 and F1 granulosa cells. However, in the presence of FSH, AICAR treatment for 36 h increased progesterone secretion, StAR protein levels and reduced extracellular signal-regulated kinase (ERK)1/2 phosphorylation in F3/4 granulosa cells. Opposite data were observed in F1 granulosa cells. Adenovirus-mediated expression of dominant-negative AMPK totally restored the effects of AICAR on FSH-induced progesterone secretion, StAR protein production, and ERK1/2 phosphorylation in F3/4 and F1 granulosa cells. Using a specific inhibitor of ERK1/2 (U0126), we also showed that this kinase is a negative regulator of the FSH-induced progesterone secretion in F3/4 and F1 granulosa cells, suggesting that AICAR-mediated AMPK activation modifies FSH-induced progesterone secretion differently through the ERK1/2 signaling pathway in hen F3/4 and F1 granulosa cells.

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

2004 ◽  
Vol 183 (1) ◽  
pp. 39-50 ◽  
Author(s):  
E C Chin ◽  
T E Harris ◽  
D R E Abayasekara
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Gradient Centrifugation ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Human Granulosa Cells ◽  
Camp Dependent Protein Kinase ◽  
Progesterone Secretion ◽  
Dependent Protein ◽  
Dose Dependent

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)IIα/catalytic (C)α expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RIIα/Cα 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 RIIα and Cα 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 PKA-independent mechanisms.

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro

1994 ◽  
Vol 143 (1) ◽  
pp. 157-164 ◽  
Author(s):  
J G Gong ◽  
D McBride ◽  
T A Bramley ◽  
R Webb
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Serum Free ◽  
Factor I ◽  
Igf I ◽  
Serum Free Conditions ◽  
Dose Dependent ◽  
Bovine Somatotrophin ◽  
Cells Cultured

Abstract Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo. Journal of Endocrinology (1994) 143, 157–164

Abstract 337: Activation of Myocardial AMP-activated Protein Kinase by Metformin Prevents Progression of Heart Failure in Dogs; Involvement of eNOS Activation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Hideyuki Sasaki ◽  
Hiroshi Asanuma ◽  
Masashi Fujita ◽  
Hiroyuki Takahama ◽  
Masanori Asakura ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Heart Failure ◽  
Cell Death ◽  
Protein Kinase ◽  
Left Ventricular ◽  
Vehicle Group ◽  
Mrna Levels ◽  
Compound C ◽  
Amp Activated Protein Kinase

Background; Several studies have shown that metformin activates AMP-activated protein kinase (AMPK), which mediates potent cardioprotection against ischemia-reperfusion injury. AMPK is also activated in experimental failing myocardium, suggesting that activation of AMPK is beneficial for the pathophysiology of heart failure. We investigated whether metformin prevents oxidative stress-induced cell death in rat cardiomyocytes and attenuates the progression of heart failure in dogs. Methods and Results; The treatment with metformin (10 μmol/L) protected the rat cultured cardiomyocytes against cell death due to H 2 O 2 exposure (50 μmol/L) as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), TUNEL staining, and flow cytometry. These effects were blunted by an AMPK inhibitor, compound-C (20 μmol/L), suggesting that the activation of AMPK decreased the extent of apoptosis-induced cell death due to H 2 O 2 exposure. Continuous rapid ventricular pacing (230/min for 4 weeks) in dogs caused heart failure and the treatment with metformin (100 mg/kg/day PO, n=8) decreased left ventricular (LV) end-diastolic dimension (32.8±0.4 vs. 36.5±1.0 mm, p< 0.01) and pressure (11.8±1.1 vs. 22±0.9 mmHg, p< 0.01), and increased LV fractional shortening (18.6±1.8 vs. 9.6±0.7 %, p< 0.01) along with enhanced phosphorylation of AMPK and the decreased the number of TUNEL-positive cells of the LV myocardium compared with the vehicle group (n=8). Interestingly, metformin increased the protein and mRNA levels of endothelial nitric oxide synthase of the LV myocardium and plasma nitric oxide levels. Metformin improved the plasma insulin resistance without increased myocardial GLUT-4 translocation. Furthermore, the subcutaneous administration of AICAR (50 mg/kg/every other day), another AMPK activator mediated the equivalent effects to metformin, strengthening the pivotal role of AMPK in reduction of apoptosis and prevention of heart failure. Conclusions; Activation of myocardial AMPK attenuated the oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with eNOS activation. Thus, metformin or AICAR may be applicable as a novel therapy for heart failure.

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

2001 ◽  
Vol 280 (5) ◽  
pp. E677-E684 ◽  
Author(s):  
Nicolas Musi ◽  
Tatsuya Hayashi ◽  
Nobuharu Fujii ◽  
Michael F. Hirshman ◽  
Lee A. Witters ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Muscle Contractions ◽  
Treadmill Running ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-β-d-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPKα1 and AMPKα2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-β-d-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3- O-methyl-d-glucose (3-MG) uptake. There were dose-dependent increases in AMPKα2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPKα1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPKα2 activity and 3-MG uptake but had little effect on AMPKα1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPKα1 and -α2 activity and 3-MG uptake. Although the AMPKα1 and -α2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPKα2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.

scholarly journals Characterization of the Role of AMP-Activated Protein Kinase in the Regulation of Glucose-Activated Gene Expression Using Constitutively Active and Dominant Negative Forms of the Kinase

2000 ◽  
Vol 20 (18) ◽  
pp. 6704-6711 ◽  
Author(s):  
Angela Woods ◽  
Dalila Azzout-Marniche ◽  
Marc Foretz ◽  
Silvie C. Stein ◽  
Patricia Lemarchand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Fatty Acid Synthase ◽  
Physiological Role ◽  
Rat Hepatocytes ◽  
Dominant Negative ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Constitutively Active

ABSTRACT In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (α1312) acts as a constitutively active kinase, while the other (α1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of α1312 increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of α1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells

2003 ◽  
Vol 284 (5) ◽  
pp. C1297-C1308 ◽  
Author(s):  
Kenneth R. Hallows ◽  
Gary P. Kobinger ◽  
James M. Wilson ◽  
Lee A. Witters ◽  
J. Kevin Foskett
Keyword(s):  
Protein Kinase ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Dominant Negative ◽  
T84 Cells ◽  
Physiological Relevance ◽  
Polarized Epithelia ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-gated Cl− channel and cellular conductance regulator, but the detailed mechanisms of CFTR regulation and its regulation of other transport proteins remain obscure. We previously identified the metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and inhibited CFTR-dependent whole cell conductances when coexpressed with CFTR in Xenopus oocytes. To address the physiological relevance of the CFTR-AMPK interaction, we have now studied polarized epithelia and have evaluated the localization of endogenous AMPK and CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical distribution in several rat epithelial tissues, including nasopharynx, submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit currents ( Isc ) were measured in polarized T84 cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog 5-amino-4-imidazolecarboxamide-1-β-d-ribofuranoside (AICAR) inhibited forskolin-stimulated CFTR-dependent I sc in nonpermeabilized monolayers and monolayers with nystatin permeabilization of the basolateral membrane. Raising intracellular AMP concentration in monolayers with basolateral membranes permeabilized with α-toxin also inhibited CFTR, an effect that was unrelated to adenosine receptors. Finally, overexpression of a kinase-dead mutant AMPK-α1 subunit (α1-K45R) enhanced forskolin-stimulated I sc in polarized T84 monolayers, consistent with a dominant-negative reduction in the inhibition of CFTR by endogenous AMPK. These results indicate that AMPK plays a physiological role in modulating CFTR activity in polarized epithelia and suggest a novel paradigm for the coupling of ion transport to cellular metabolism.

scholarly journals Sex Differences in the Metabolic Effects of Testosterone in Sheep

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 123-131 ◽  
Author(s):  
Scott D. Clarke ◽  
Iain J. Clarke ◽  
Alexandra Rao ◽  
Michael A. Cowley ◽  
Belinda A. Henry
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Uncoupling Protein ◽  
Specific Effect ◽  
Metabolic Effects ◽  
Mrna Levels ◽  
Nonesterified Fatty Acids ◽  
Glucose Levels ◽  
Amp Activated Protein Kinase ◽  
The Brain

Adiposity is regulated in a sexually divergent manner. This is partly due to sex steroids, but the differential effects of androgens in males and females are unclear. We investigated effects of testosterone on energy balance in castrated male (n = 6) and female sheep (n = 4), which received 3 × 200 mg testosterone implants for 2 wk or blank implants (controls). Temperature probes were implanted into retroperitoneal fat and skeletal muscle. Blood samples were taken to measure metabolites and insulin. In males, muscle and fat biopsies were collected to measure uncoupling protein (UCP) mRNA and phosphorylation of AMP-activated protein kinase and Akt. Testosterone did not change food intake in either sex. Temperature in muscle was higher in males than females, and testosterone reduced heat production in males only. In fat, however, temperature was higher in the castrate males compared with females, and there was no effect of testosterone treatment in either sex. Preprandial glucose levels were lower, but nonesterified fatty acids were higher in females compared with males, irrespective of testosterone. In males, the onset of feeding increased UCP1 and UCP3 mRNA levels in skeletal muscle, without an effect of testosterone. During feeding, testosterone reduced glucose levels in males only but did not alter the phosphorylation of AMP-activated protein kinase or Akt in muscle. Thus, testosterone maintains lower muscle and fat temperatures in males but not females. The mechanism underlying this sex-specific effect of testosterone is unknown but may be due to sexual differentiation of the brain centers controlling energy expenditure.

Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

1999 ◽  
Vol 276 (2) ◽  
pp. G322-G330 ◽  
Author(s):  
Brian K. Dieckgraefe ◽  
Danielle M. Weems
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Erk Activation ◽  
Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

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