Short-Term Modulation of Extracellular Signal-Regulated Kinase 1/2 and Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in Pancreatic Islets by Glucose and Palmitate Possible Involvement of Ceramide

Objectives: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. Methods: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. Results: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. Conclusions: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK. gene transcription.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

RECK-Mediated Inhibition of Glioma Migration and Invasion

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDKI and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors. (C) 2004 Elsevier B.V. All rights reserved.

In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Osteoblast Adhesion Dynamics: A Possible Role for ROS and LMW-PTP

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

TNF alpha contributes for attenuating both Y(397)FAK and Y(416)Src phosphorylations in osteoblasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (similar to 998 angstrom(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.