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Melanocytes play a major role in the formation of mammalian fur color and are regulated by several genes. Despite playing the pivotal role in the study of melanoma, the mechanistic role of NRAS (neuroblastoma RAS viral oncogene homolog) in the formation of mammalian epidermal color is still elusive. First of all, the expression levels of NRAS mRNA and protein in the dorsal skin of different colored Rex rabbits were detected by qRT-PCR and Western blot. Then, the subcellular localization of NRAS was identified in melanocytes by indirect immunofluorescence. Next, the expression of NRAS was overexpressed and knocked down in melanocytes, and its efficiency was verified by qRT-PCR and Western blot. Subsequently, NaOH, CCK-8, and Annexin V-FITC were used to verify the changes in melanin content, proliferation, and apoptosis in melanocytes. Finally, we analyzed the regulation of NRAS on other genes (MITF, TYR, DCT, PMEL, and CREB) that affect melanin production. In silico studies showed NRAS as a stable and hydrophilic protein, and it is localized in the cytoplasm and nucleus of melanocytes. The mRNA and protein expression levels of NRAS were significantly different in skin of different colored Rex rabbits, and the highest level was found in black skin (P < 0.01). Moreover, the NRAS demonstrated impact on the proliferation, apoptosis, and melanin production of melanocytes (P < 0.05), and the strong correlation of NRAS with melanin-related genes was evidently observed (P < 0.05). Our results suggested that NRAS can be used as a gene that regulates melanin production and controls melanocyte proliferation and apoptosis, providing a new theoretical basis for studying the mechanism of mammalian fur color formation. Key words: NRAS, melanocytes, melanin, Rex rabbit, melanin-related genes, Introduction Melanin is a biological pigment synthesized by melanocytes, which is transported outside the cells and deposited in various tissues such as hair, skin, and eyes through a specific transport [...]

A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creER.sup.T2; Lepr.sup.fl/fl mice reveals that leptin's effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes. The beneficial effects of exercise on cardiovascular disease are linked to decreased inflammation through crosstalk between adipose tissue and hematopoietic progenitor cells in the bone marrow., Author(s): Vanessa Frodermann [sup.1] , David Rohde [sup.1] , Gabriel Courties [sup.1] , Nicolas Severe [sup.2] [sup.3] , Maximilian J. Schloss [sup.1] , Hajera Amatullah [sup.4] , Cameron S. McAlpine [...]

Abstract: Aberrant DNA methylation has been shown toinactivate tumor suppressor genes during carcinogenesis. MicroRNA-149 (miR-149) was recently demonstrated to function as a tumor suppressor gene in glioblastoma multiforme (GBM). However, [...]

Abstract: Hepatocyte proliferation and collagen I (COLI) secretion are important processes during liver regeneration. This study aimed to investigate the role of CXCL6 in hepatocyte proliferation and COLI secretion. Serum [...]

ABSTRACT Background: Cancer is one of the leading causes of death worldwide, both in developed and developing countries. Of note, colorectal adenoma encompasses a high rate of gastrointestinal-associated cancer death [...]

Abstract: MicroRNAs (miRNAs) are novel and potent regulators in myogenesis. However, the molecular mechanisms that many miRNAs regulate myoblast proliferation and differentiation which are largely unknown. Here, we found that [...]

Author(s): Alexandra C Schmiechen [1]; Pedro J Batista (corresponding author) [1] All blood cell types derive from a common progenitor, the hematopoietic stem cell, through a hierarchical process. In one [...]

Fat deposition is a complex process involving proliferation, differentiation, and lipogenesis of adipocytes. Bamei and Landrace are considered to represent fat- and lean-type pig breeds. Subcutaneous (SC) and intramuscular (IM) pre- adipocytes were cultured to compare the proliferation and lipogenesis in these breeds. The differentiated adipocytes were exposed to glucose or insulin to evaluate their effects on lipogenesis and lipogenic gene expression. Pre-adipocytes proliferated dramatically faster in SC vs. IM cells, and in Bamei vs. Landrace breeds. Lipogenesis and lipogenic gene expression had a greater increase in Bamei than in Landrace, and in SC vs. IM in the process of differentiation. Glucose markedly promoted lipogenesis and lipogenic gene expression in differentiated adipocytes. The stimulation of high-glucose levels on lipogenesis and ChREBP and lipogenic gene expression was higher in SC than IM adipocytes, and in Bamei vs. Landrace. Insulin largely increased SREBP-lc expression, however it modestly stimulated lipogenesis and lipogenic gene expression, and there was no difference between cell populationsor between breeds. These data demonstrated that regional and varietal differences obviously existed in the development of porcine adipocytes. The proliferation and differentiation capacity of pre-adipocytes, and the adipocyte lipogenesis stimulated by glucose, are stronger in Bamei than Landrace, and in SC vs. IM adipocytes independent of breed. Key words: Bamei pigs, intramuscular adipocytes, subcutaneous adipocytes, proliferation, adipogenesis. Le depot de graisse est un processus complexe qui implique la proliferation, la differenciation et la lipogenese des adipocytes. On considere que les races procines Bamei et Landrace sont representatives des races de type grasse ou maigre. Des pre-adipocytes SC et IM ont ete cultives afin de comparer la proliferation et la lipogenese chez ces deux races. Les adipocytes differencies ont ete exposes au glucose ou a l'insuline afin d'evaluer leurs effets sur la lipogenese et l'expression de genes lies a lipogenese. Les pre-adipocytes SC proliferaient beaucoup plus vite que les pre-adipocytes IM, et davantage chez le Bamei que le Landrace. La lipogenese et l'expression de genes lies a la lipogenese etaient plus elevees chez le Bamei comparativement au Landrace, et chez les SC comparativement aux IM lors de la differenciation. Le glucose favorisait de maniere importante la lipogenese et l'expression de genes lies a la lipogenese chez les adipocytes differencies. La stimulation de la lipogenese et de l'expression de ChREBP et de genes lies a la lipogenese par des doses elevees de glucose etait plus elevee chez les adipocytes SC comparativement aux IM, et chez le Bamei comparativement au Landrace. L'insuline augmentait grandement l'expression de SREBP-lc, mais stimulait de fa^on modeste la lipogenese et l'expression de genes lies a la lipogenese, et il n'y avait pas de difference entre les populations cellulaires et entre les races. Ces donnees demontrent que les differences de localisation et de race existent de maniere evidente dans le developpement des adipocytes chez le porc. Les capacites de proliferation et de differenciation des pre-adipocytes, et la lipogenese stimulee par le glucose sont plus elevees chez le Bamei comparativement au Landrace, et chez les adipocytes SC comparativement aux IM, independamment de la race. [Traduit par la Redaction] Mots-cles: porcs Bamei, adipocytes intramusculaires, adipocytes sous-cutanes, proliferation, adipogenese., Introduction Pig breeding during recent decades has focused on improving growth and muscularity. The selection direction results from efforts to eliminate the consequences of long-term marginalization of fatness traits due [...]

Although myostatin functions primarily as a negative regulator of skeletal muscle growth and development, accumulating biological and epidemiological evidence indicates an important contributing role in liver disease. In this study, we demonstrate that myostatin suppresses the proliferation of mouse Hepa-1c1c7 murine-derived liver cells (50%; p < 0.001) in part by reducing the expression of the cyclins and cyclin-dependent kinases that elicit G1-S phase transition of the cell cycle (p Key words: hepatogenesis, glucose metabolism, myostatin, Malatl siRNA, cell cycle. Meme si la myostatine agit d'abord comme regulateur negatif de la croissance et du developpement du muscle squelettique, il existe de plus en plus de preuves sur le plan de la biologie et de l'epidemiologie qui indiquent qu'elle contribute de maniere importante aux maladies hepatiques. Dans cette etude, les auteurs demontrent que la myostatine supprime la proliferation des cellules Hepa-1c1c7 derivees du foie de souris (50%; p < 0.001), en partie en reduisant l'expression des cyclones et des kinases dependantes des cyclines qui declenchent la transition G1-S du cycle cellulaire (p < 0.001). De plus la quantification par RT-PCR du long ARN non codant Malat1, identifie recemment comme transcrit repondant a la myostatine dans le muscle lisse, a revele qu'il etait regule a labaisse dans les foies des souris et dans les cellules de foie traitees a la myostatine (25% et 50% respectivement, p < 0.05). L'importance de Malat1 dans la proliferation des cellules de foie a ete confirmee par un arret de la proliferation des cellules hepatiques (p < 0.05) en reponse a un knockdown de Malat1 par pARNi. La myostatine attenuait aussi significativement la captation de glucose stimulee par l'insuline et la phosphorylation d'Akt dans les cellules de foie, alors qu'elle augmentait la phosphorylation de MARCKS (Myristoylated Alanine-Rich Kinase C Substrate), une proteine essentielle a la proliferation des cellules cancereuses et au transport du glucose stimule par l'insuline. Dans leur ensemble, ces donnees revelent un mecanisme plausible par lequel la myostatine circulante contribue a la diminution de la capacite regenerative du foie et aux maladies caracterisees par une resistance du foie a l'insuline. [Traduit par la Redaction] Mots-cles: hepatogenese, metabolisme du glucose, myostatine, pARNi de Malat1, cycle cellulaire., Introduction Myostatin is a secreted member of the transforming growth factor-β (TGF-β) family that acts as a negative regulator of skeletal muscle growth and development (Rios et al. 2004) by [...]

Aims: To investigate whether ginsenoside Rg1 can promote neural phenotype differentiation of human adipose-derived stem cells (hASCs) in vitro. Methods: hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3rd passage. Cultured hASCs were treated with neural inductive media alone (group A, control) or inductive media plus 10, 50, or 100 pg/mL ginsenoside Rg1 (groups B, C, and D, respectively). Cell proliferation was assessed by CCK-8 assay. Neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2) levels were measured by Western blot. mRNA levels of growth associated protein-43 (GAP-43), neural cell adhesion molecule (NCAM), and synapsin-1 (SYN-1) were determined by real-time PCR. Results: Ginsenoside Rg1 promoted the proliferation of hASCs (groups B, C, and D) and resulted in higher expression of NSE and MAP-2 compared with the control group. Gene expression levels of GAP- 43, NCAM, and SYN-1 in the test groups were higher than that in thw control. The results displayed a dose-dependent effect of ginsenoside Rg1 on cell proliferation and neural phenotype differentiation. Conclusion: This study indicated that ginsenoside Rg1 promotes cell proliferation and neural phenotype differentiation of hASCs in vitro, suggesting a potential use for hASCs in neural regeneration medicine. Key words: adipose-derived stem cells, cell therapy, induced differentiation, neural regeneration, ginsenoside Rg1, cell proliferation, neural phenotype. Buts : Examiner si le ginsenoside Rg1 peut promouvoir la differenciation des cellules souches humaines derivees du tissu adipeux (ou hASC pour human adipose-derived stem cells) vers un phenotype neural in vitro. Methodes : les hASC ont ete isolees a partir de lipo-aspirations et caracterisees a l'aide de marqueurs cellulaires specifiques et selon leur capacite de differenciation en lignages multiples apres une culture jusqu'au 3ieme passage. Les hACS en culture ont ete traitees avec un milieu induisant un phenotype neural (groupe A, controle), ou un milieu inducteur comprenant 10,50, ou 100 pg/mL de ginsenoside Rg1 (groupes B, C et D, respectivement). La proliferation cellulaire a ete evaluee a l'aide d'un dosage CCK-8. Les niveaux d'enolase specifique au neurone NSE et de la proteine associee aux microtubules MAP-2 ont ete mesures par buvardage Western. Les niveaux d'ARNm de la proteine GAP-43, de la molecule d'adhesion NCAM et de la synapsine-1 (SYN-1) ont ete determines par PCR en temps reel. Resultats : Le ginsenoside Rg1 pouvait promouvoir la proliferation des hASC (groupes B, C, et D), ce qui resultait en une expression accrue de NSE et de MAP-2 comparativement au groupe controle. Les niveaux d'expression genique de GAP-43, NCAM et SYN-1 dans les groupes test etaient superieurs a ceux du groupe controle. Les resultats montraient un effet dependant de la concentration du ginsenoside Rg1 sur la proliferation cellulaire et la differenciation vers un phenotype neural. Conclusion : Cette etude a indique que le ginsenoside Rg1 peut promouvoir la proliferation et la differenciation des hASC vers un phenotype neural in vitro, suggerant que les hASC pourraient possiblement etre utilisees en neurologie regenerative. [Traduit par la Redaction] Mots-cles: cellules souches derivees du tissu adipeux, therapie cellulaire, differenciation induite, regeneration neurale, ginsenoside Rg1, proliferation cellulaire, phenotype neural., Introduction Injuries to the nervous system are very common and represent a major health problem worldwide. It has been estimated that approximately 90 000 people suffer from such conditions every [...]

Exosomes, which are small membrane vesicles of endocytic origin, carry lipids, RNA/miRNAs, and proteins and have immune modulatory functions. In this study, we isolated exosomes from the bile of specific pathogen-free chickens, 42- 43 days of age, by using an ultracentrifugation and filtration method. The density of the exosomes, isolated by sucrose gradient fractionation, was between 1.13 and 1.19 g/mL. Electron microscopic observation of the liver showed that exosomes were present in the space of Disse and bile canaliculus. Chicken biliary exosomes displayed typical saucer-shaped, rounded morphology. Using liquid chromatography mass spectrum methodology, 196 proteins, including exosomal markers and several unique proteins, were identified and compared with mouse biliary exosomes. Noteworthy, CCCH type zinc finger antiviral protein was found on chicken biliary exosomes never described before. Furthermore, our data show that chicken biliary exosomes promote the proliferation of [CD4.sup.+] and [CD8.sup.+] T cells and monocytes from liver. In addition, chicken biliary exosomes significantly inhibit avian leukosis virus subgroup J, which is an oncogenic retrovirus, from replicating in the DF-1 cell line. These data indicate that chicken biliary exosomes possess the capacity to influence the immune responses of lymphocytes and inhibit avian leukosis virus subgroup J (ALV-J). Key words: chicken biliary exosomes, zinc finger antiviral protein, liver, [CD4.sup.+]T, ALV-J. Les exosomes, des petites vesicules membranaires d'origine endocytique, transportent des lipides, de l'ARN/miARN et des proteines, et elles exercent des fonctions modulatrices de la reponse immune. Dans cette etude, nous avons isole des exosomes de la bile de poulets depourvus de pathogenes specifiques, ages de 42-43 jours, en utilisant l'ultracentifugation et une methode de filtration. La densite des exosomes isoles par fractionnement sur gradient de sucrose se situait entre 1.13 et 1.19 g/mL. L'observation du foie en microscopie electronique a montre que les exosomes etaient presents dans l'espace de Disse et les canalicules biliaires. Les exosomes biliaires de poulet presentaient une morphologie arrondie typique, en forme de soucoupe. A l'aide de la chomatographie liquide couplee a la spectrometrie de masse, 196 proteines, incluant les marqueurs exosomaux et plusieurs proteines uniques, ont ete identifiees comparativement aux exosomes biliaires de souris. Fait important, la proteine antivirale a doigt de zinc de type CCCH etait trouvee dans les exosomes biliaires de poulet, ce qui n'avait jamais ete decrit auparavant. De plus, nos donnees montrent que les exosomes biliaires de poulet favorisent la proliferation des cellules T [CD4.sup.+] et [CD8.sup.+], ainsi que des monocytes du foie. En outre, les exosomes biliaires de poulet inhibent significativement le virus de la leucose aviaire du sous-groupe J, un retrovirus oncogene, a partir de la lignee DF-1 en replication. Ces donnees indiquent que les exosomes biliaires de poulet possedent la capacite d'influencer les reponses immunes des lymphocytes et d'inhiber le virus de la leucose aviaire du sous-groupe J (VLA-J). [Traduit par la Redaction] Mots-cles: exosomes biliaires de poulet, proteine antivirale a doigt de zinc, foie, cellules T [CD4.sup.+], VLA-J., Introduction Exosomes are 30-100 nm membrane vesicles of endocytic origin secreted by various cell types (Admyre et al. 2007; Conde-Vancells et al. 2008; Witek et al. 2009; Lasser et al. [...]

Septins are cytoskeletal proteins that form hetero-oligomeric complexes and function in many biological processes, including cytokinesis. Drosophila melanogaster has five septin genes. Sep5, which is the most recently evolved septin gene in Drosophila, is a retrogene copy of Sep2. Sep5 mutants appear wild type, whereas Sep2 mutant females are semisterile. Their ovaries have egg chambers containing abnormal numbers of nurse cells. The egg chamber phenotype is rescued to wild type by expressing a Sep2 cDNA, but it is only partially rescued by expressing a Sep5 cDNA, showing that these paralogs have diverged in function at the protein level. Sep2 Sep5 double mutants have an early pupal lethal phenotype and lack imaginal discs, suggesting that these genes have redundant functions during imaginal cell proliferation. Key words: gene duplication, septins, oogenesis, synthetic lethality, retrogene. Les septines sont des proteines du cytosquelette qui forment des complexes hetero-oligomeriques et elles sont impliquees dans plusieurs processus biologiques comme la cytocinese. Le Drosophila melanogaster possede cinq genes codant pour des septines. Sep5, le gene de septine le plus recent au plan evolutif chez la drosophile, est une copie retrogene de Sep2. Les mutants pour Sep5 presentent un phenotype sauvage, tandis que les femelles des mutants Sep2 sont semi-steriles. Leurs ovaires contiennent des chambres a ceuf avec un nombre anormal de cellules nourricieres. Le phenotype de la chambre a ceuf est ramene a l'etat normal en exprimant l'ADNc de Sep2, mais il est seulement partiellement retabli en exprimant l'ADNc de Sep5,cequi montre que ces paralogues ont diverge quant a la fonction de ces proteines. Les doubles mutants Sep2 Sep5 presentent une letalite tot au cours du developpement de la pupe et sont depourvus de disques imaginaux, ce qui suggere que ces genes sont fonctionnellement redondants au cours de la proliferation des cellules imaginales. [Traduit par la Redaction] Mots-cles: duplication genique, septines, oogenese, letalite synthetique, retrogene., Introduction Septins were first identified in Saccharomyces cerevisiae as temperature-sensitive mutants defective in cytokinesis (Hartwell et al. 1970; Hartwell 1971; Longtine et al. 1996). They can assemble into hetero-oligomeric complexes, [...]

This study evaluated the in vitro expression of bone-related proteins by osteoblasts in the presence of different concentrations of human recombinant bone morphogenetic protein-2 (rhBMP-2). Immortalized human fetal osteoblastic cell line 1.19 (hFOB) were exposed to different concentrations of rhBMP-2 (10,50, or 100 ng/mL) for 72 h. Cell proliferation and viability (MTT assay), as well as the expression of fibronectin, osteonectin, and osteopontin were assessed by indirect immunofluorescence and Western blot. Neither of the 3 concentrations of rhBMP-2 caused statistically significant alterations in cell proliferation and viability, although the concentration of 100 ng/mL showed lower values for both assays after both 48 and 72 h of exposure. There was no alteration in the expression of noncollagenous proteins, as analyzed by immunofluorescence, when compared with the control group. Furthermore, in the Western blot assay we observed a statistically significant decrease in fibronectin and osteonectin at 100 ng rhBMP-2/mL (p < 0.05) by comparison with the medium alone. The expression of osteopontin decreased slightly in all 3 concentrations of rhBMP-2 tested; however, the change was not statistically significant (p > 0.05). In this in-vitro study, the tested concentrations of rhBMP-2 appeared to decrease the expression of important bone-related molecules in pre-osteoblast cells. Key words: bone, bone morphogenetic protein, osteoblast. L'etude presente a evalue l'expression de proteines osseuses par les osteoblastes in vitro en presence de differentes concentrations de BMP-2 humain recombinant (rhBMP-2, human recombinant bone morphogenetic protein-2). La lignee cellulaire d'osteoblastes foetaux immortalises 1.19 (hFOB) a ete exposee a differentes concentrations de rh-BMP-2 (10, 50 et 100 ng/mL) pendant 72 h. La proliferation et la viabilite cellulaires (dosage MTT) de meme que l'expression de la fibronectine, de l'osteonectine et de l'osteopontine ont ete evaluees par immunofluorescence indirecte et par buvardage Western. Les 3 differentes concentrations de rh-BMP-2 n'induisaient aucune modification statistiquement significative de la proliferation et la viabilite cellulaires, quoiqu'a une concentration de 100 ng/mL, les deux tests presentaient des valeurs plus faibles apres 48 et 72 h. Aucune modification de l'expression des proteines non collageniques n'etait observee en immunofluorescence comparativement au groupe controle. En outre, une diminution statistiquement significative de la fibronectine et de l'osteonectine etait observee par buvardage Western a 100 ng/mL (p < 0,05) comparativement au milieu seul. L'expression de l'osteopontine etait legerement diminuee par les trois concentrations testees, toutefois, la difference n'etait pas statistiquement significative. Dans cette etude realisee in vitro, les concentrations de rhBMP-2 testees semblaient diminuer l'expression d'importantes molecules reliees a l'os chez les pre-osteoblastes. [Traduit par la Redaction] Mots-cles: os, proteine morphogenetique de l'os, osteoblaste., Introduction Bony defects caused by pathology, trauma, infection, and oncologic resections can be restored using several surgical procedures. The currently available sources of bone are from the host (autograft, considered [...]

ARTICLE INFO Keywords: Antrodia cinnamomea Mushroom WEHI-3 cells Migration Proliferation Antileukemic ABSTRACT The aim of this study was to explore whether the ethanolic extract of Antrodia cinnamomea (EEAC), a medical [...]

ARTICLE INFO Keywords: Puerarin Proliferation Differentiation Survival Osteoblast ABSTRACT Puerarin, the main isoflavone glycoside found in the Chinese herb radix of Pueraria lobata (Willd.) Ohwi, has received increasing attention because [...]

ARTICLE INFO Keywords: Curcuma comosa Diarylheptanoid ERK 1/2 Osteoporosis Phytoestrogen Selective estrogen receptor modulator ABSTRACT Curcuma comosa Roxb, is ginger-family plant used to relieve menopausal symptoms. Previous work showed that [...]

Insulin-like growth factor 1 (IGF-1) is a mitogenic factor that stimulates the signaling pathways responsible for inducing hypertrophic and proliferative responses in vascular smooth muscle cells (VSMC). We have previously demonstrated that IGF-1 receptor (IGF-1R) plays a key role in transducing the hypertrophic and proliferative responses of angiotensin II (Ang-II) and endothelin-1 (ET-1). Curcumin, a polyphenolic compound derived from the spice turmeric is known to possess antiproliferative properties and exerts vasculoprotective effects. However, the ability of curcumin to modulate IGF-1-induced signaling responses in VSMC remains to be investigated. In this study, we determined the effect of curcumin on IGF-1-induced phosphorylation of protein kinase B (PKB), glycogen synthase kinase-3β (GSK-3β), and IGF-1R in VSMC. Curcumin inhibited IGF-1-induced phosphorylation of PKB and GSK-3β as well as the IGF-1R β subunit in a dose-dependent fashion. In addition, IGF-1-induced expression of early growth response protein 1 (Egr-1) which plays a pathogenic role in vascular dysfunctions, was also attenuated by curcumin. In conclusion, these results indicate that curcumin is a potent inhibitor of key components of the IGF-1-induced mitogenic and proliferative signaling system in VSMC, and suggest that curcumin-induced attenuation of these signaling components may constitute a potential mechanism for its vasculoprotective effects. Keywords: curcumin, insulin-like growth factor-1, protein kinase B, glycogen synthase kinase-3β, early growth response protein-1, vascular smooth muscle cells., L'IGF-1 (Insulin-like growth factor-1) est un facteur mitogene qui stimule les voies de signalisation responsables de l'induction des reponses hypertrophique et proliferative chez les cellules musculaires lisses vasculaires (CMLV). Nous [...]

We have recently shown that vasoactive peptides such as angiotensin II (Ang II) and endothelin-1 (ET-1) increase the expression of Gi proteins and the proliferation of A10 vascular smooth muscle cells (VSMC) through mitogen-activated protein (MAP) kinase--phosphoinositide (PI) 3-kinase pathways. This study was intended to examine the implication of epidermal growth factor receptor (EGFR) activation in ET-1-induced enhanced expression of Gi proteins and proliferation of A10 VSMC, and to further investigate the underlying mechanisms responsible for these increases. Cell proliferation was determined by [³thymidine incorporation and the expression of Gi proteins; extracellular signal-regulated kinases 1 and 2 (ERK1/2) and EGFR phosphorylation was determined by Western blotting. Treatment of A10 VSMC with ET-1 enhanced the expression of Gi proteins, which was attenuated by BQ123 and BQ788, antagonists of [ET.sub.A] and [ET.sub.B] receptor respectively. In addition, ET-1 enhanced the phosphorylation of EGFR in A10 VSMC, which was restored to the control levels by EGFR inhibitor and [ET.sub.A] and [ET.sub.B] receptor antagonists. Furthermore, ET-1 also augmented the proliferation and ERK1/2 phosphorylation of A10 VSMC, which were restored to the control levels by inhibition of EGFR. These data suggest that ET-1 transactivates EGFR, which, through MAP kinase signaling, may contribute to the enhanced expression of Gi proteins and thus increased proliferation of A10 VSMC. Key words: ET-1, EGFR, ERK1/2, Gi proteins, VSMC proliferation. Nous avons recemment montre que les peptides vasoactifs comme l'angiotensine II (Ang II) et l'endotheline-1 (ET-1) augmentent l'expression des proteines Gi et la proliferation des cellules musculaires lisses vasculaires (CMLV) par l'intermediaire des voies MAP kinase--PI 3-kinase. La presente etude a ete realisee afin d'examiner l'implication de l'activation du recepteur de l'EGF (EGFR) dans l'augmentation de l'expression des proteines Gi et de la proliferation des CMLV A10 induites par l'ET-1, et d'examiner plus en profondeur les mecanismes sous-jacents responsables de ces augmentations. La proliferation cellulaire a ete determinee par incorporation de [³H]thymidine, et l'expression des proteines Gi ainsi que la phosphorylation de ERK1/2 et du EGFR ont ete determinees par buvardage Western. Le traitement de cellules A10 avec l'ET-1 augmentait l'expression des proteines Gi, laquelle etait attenuee par le BQ123 et le BQ788, des antagonistes des recepteurs [ET.sub.A] et [E.sub.TB] respectivement. En outre, l'ET-1 augmentait la phosphorylation du EGFR chez les cellules A10, laquelle etait ramenee aux niveaux controles par un inhibiteur du EGFR et par les antagonistes des recepteurs [ET.sub.A] et [ET.sub.B]. De plus, l'ET-1 augmentait aussi la proliferation et la phosphorylation de ERK1/2 chez les cellules A10, lesquelles etaient ramenees aux niveaux controles par un inhibiteur du EGFR. Ces donnees suggerent que l'ET-1 trans-active le EGFR qui, par l'intermediaire de la signalisation par les MAP kinases, peut contribuer a l'augmentation de l'expression des proteines Gi et consequemment, stimuler la proliferation des cellules A10. [Traduit par la Redaction] Mots-cles: ET-1, EGFR, ERK1/2, proteines Gi, proliferation des CMLV., Introduction Guanine-nucleotide-binding regulatory proteins (G proteins), a family of GTP-binding proteins play an important role in the regulation of a variety of physiological functions, including blood pressure, through the activation [...]

Mechanical stress contributes to hypertension and atherosclerosis partly through the stimulation of vascular smooth muscle cell (VSMC) proliferation. Oxidized low density lipoprotein (oxLDL) is another important atherogenic factor that can increase VSMC proliferation. The purpose of this study was to investigate whether oxLDL could further enhance the proliferative action of mechanical stretch on VSMC, and to determine the mechanism responsible for this interaction. Because nuclear protein import is critical in regulating gene expression, transcription, and cell proliferation, its involvement in the mitogenic effects of oxLDL and mechanical stress was studied. OxLDL enhanced the proliferative effects of mechanical stretch on its own in rabbit aortic VSMC, and induced increases in the expression of HSP60 in an additive manner. Adenoviral-mediated overexpression of HSP60 induced increases in cell proliferation compared with uninfected VSMC. Mechanical stretch and oxLDL stimulated the rate of nuclear protein import in VSMC and increased the expression of nucleoporins. These effects were sensitive to inhibition of the MAPK pathway. We conclude that oxLDL and mechanical stretch have a synergistic effect on VSMC proliferation. This synergistic effect is induced through a stimulation of nuclear protein import via HSP60 and an activation of the MAPK pathway. Key words: mechanical stretch, oxidized low-density lipoprotein, heat shock protein, mitogen activated protein kinase, cell proliferation. Le stress mecanique contribue a l'hypertension et l'atherosclerose en partie par l'intermediaire de la stimulation de la proliferation des cellules de muscle lisse vasculaire (CMLV). Les lipoproteines de faible densite oxydees (oxLDL) sont egalement d'importants facteurs atherogenes qui peuvent aussi augmenter la proliferation de CMVL. Le but de la presente etude etait d'examiner si les oxLDL peuvent accroitre l'action proliferative de l'etirement mecanique sur les CMVL et de determiner le mecanisme responsable de cette interaction. Parce que l'import nucleaire des proteines est determinant dans la regulation de l'expression genique, la transcription et la proliferation cellulaire, son implication dans les effets mitogenes des oxLDL et du stress mecanique a ete etudiee. Les oxLDL augmentaient les effets proliferatifs de l'etirement mecanique sur les CMVL d'aorte de lapin et induisait une augmentation de l'expression de HSP60 de maniere additive. La surexpression de HSP60 par adenovirus induisait une augmentation de la proliferation cellulaire comparativement aux CMLV non infectees. L'etirement mecanique et les oxLDL stimulaient le taux d'import nucleaire chez les CMLV et augmentaient l'expression des nucleoporines. Ces effets etaient sensibles a l'inhibition de la voie MAPK. Nous concluons que les oxLDL et l'etirement mecanique exercent un effet synergique sur la proliferation des CMLV. Cet effet synergique est induit par la stimulation de l'import nucleaire des proteines par l'intermediaire de HSP60 et d'une activation de la voie MAPK. Mots-cles: etirement mecanique, lipoproteine de faible densite oxydee, proteine de choc thermique, kinase MAPK, proliferation cellulaire. [Traduit par la Redaction], Introduction Hemodynamic forces produce shear stress, and the subsequent increase in mechanical stretch of the vessel wall can induce vascular smooth muscle cell (VSMC) proliferation, which may in turn contribute [...]

Most cardiac diseases are associated with fibrosis. Calcineurin (CaN) is regulated by [Ca.sup.2+]/calmodulin (CaM). The CaN-NFAT (nuclear factor of activated T cell) pathway is involved in the process of cardiac diseases, such as cardiac hypertrophy, but its effect on myocardial fibrosis remains unclear. The present study investigates whether the CaN-NFAT pathway is involved in cardiac fibroblast (CF) proliferation induced by electrical field stimulation (EFS), which recently became a popular treatment for heart failure and cardiac tissue engineering. CF proliferation was evaluated by a cell survival assay (MTT) and cell counts. Myocardial fibrosis was assessed by collagen I and collagen III protein expression. Green fluorescent protein (GFP)-tagged NFAT was used to detect NFAT nuclear translocation. CF proliferation, myocardial fibrosis, CaN activity, and NFAT nuclear translocation were enhanced by EFS. More importantly, these effects were abolished by CaN inhibitors, dominant negative CaN (DN-CaN), and CaN gene silenced with siRNA. Furthermore, buffering intracellular [Ca.sup.2+] with BAPTA-AM and blocking [Ca.sup.2+] influx with nifedipine suppressed EFS-induced increase in intracellular [Ca.sup.2+] and CF proliferation. These results suggested that the CaN-NFAT pathway mediates CF proliferation, and that the CaN-NFAT pathway might be a possible therapeutic target for EFS-induced myocardial fibrosis and cardiac tissue engineering. Key words: electrical field stimulation, calcineurin, nuclear factor of activated T cells, rat, cardiac fibroblasts, proliferation. La plupart des maladies cardiaques sont associees a la fibrose. La calcineurine (CaN) est regulee par la [Ca.sup.2+]/ calmoduline (CaM). La voie CaN-NFAT (nuclear factor of activated T cell) est impliquee dans le developpement de maladies cardiaques comme l'hypertrophie cardiaque, mais son effet sur la fibrose du myocarde demeure mal defini. La presente etude vise a determiner si la voie CaN/NFAT est impliquee dans la proliferation des fibroblastes cardiaques (FC) induite par une stimulation par champ electrique (SCE), qui est recemment devenue populaire comme traitement de l'insuffisance cardiaque et dans l'ingenierie cardiaque electrique. La proliferation de FC a ete evaluee par un test de survie cellulaire (MTT) et par decompte cellulaire. La fibrose du myocarde a ete evaluee par l'expression du collagene I et du collagene III. NFAT marque par une etiquette de proteine fluorescente verte GFP a ete utilise pour detecter la translocation nucleaire de NFAT. La proliferation [d.sub.es] FC, la fibrose du myocarde, l'activite de la CaN et la translocation nucleaire de NFAT et aient augmentees par la SCE. Qui plus est, ces effets etaient abolis par des inhibiteurs de CaN, par un dominant negatif de CaN (DN-CaN) et par le silencage genique de CaN par un siARN. En outre, tant le tamponnage de [Ca.sup.2+] intracellulaire par le BAPTA-AM que le bloquage d'influx de [Ca.sup.2+] par la nifedipine supprimaient l'accroissement de [Ca.sub.2+] intracellulaire induit par la SCE et la proliferation des FC. Ces resultats suggerent que la proliferation des FC dependante de la voie CaN-NFAT et CaN-NFAT pourraient possiblement constituer des cibles therapeutiques dans la fibrose du myocarde induite par la SCE et l'ingenierie cardiaque electrique. Mots-cles: stimulation par champ electrique, calcineurine, facteur NFAT, fibroblastes cardiaques, proliferation. [Traduit par la Redaction], Introduction Most cardiac diseases are reported to be associated with fibrosis (Krenning et al. 2010). In general, fibrosis is a scarring process that is characterized by fibroblast proliferation and excess [...]

A defining feature of embryonic cardiomyocytes is their relatively high rates of proliferation. A gradual reduction in proliferative capacity throughout development culminates in permanent cell cycle exit by the vast majority of cardiomyocytes around the perinatal period. Accordingly, the adult heart has severely limited capacity for regeneration in response to injury or disease. The D-type cyclins (cyclin D1, D2, and D3) along with their catalytically active partners, the cyclin dependent kinases, are positive cell cycle regulators that play important roles in regulating proliferation of cardiomyocytes during normal heart development. While expression of D-type cyclins is generally low in the adult heart, expression levels are augmented in association with cardiac hypertrophy, but are uncoupled from myocyte cell division. Accordingly, re- activation of D-type cyclin expression in the adult heart has been implicated in pathophysiological processes via mechanisms distinct from those that drive proliferation during cardiac development. Growth factors and other exogenous agents regulate D- type cyclin production and activity in embryonic and adult cardiomyocytes. Understanding differences in the precise intracellular mediators downstream from these signalling molecules in embryonic versus adult cardiomyocytes could prove valuable for designing strategies to reactivate the cell cycle in cardiomyocytes in the setting of cardiovascular disease in the adult heart. Key words: D-type cyclins, heart development, hypertrophy, cell cycle, cardiovascular disease. Les cardiomyocytes embryonnaires se caracterisent par un taux de proliferation relativement eleve. Chez la majorite des cardiomyocytes, la reduction graduelle de leur capacite proliferative au cours du developpement culmine en une sortie permanente du cycle cellulaire autour de la periode perinatale. Consequemment, le coeur adulte possede une capacite extremement limitee de regeneration en reponse a un dommage ou une maladie. Les cyclines de type D (cycline D1, D2 et D3) et leurs partenaires actifs sur le plan catalytique, les kinases dependantes des cyclines, sont des regulateurs positifs du cycle cellulaire qui jouent un role important en regulant la proliferation des cardiomyocytes durant le developpement normal du coeur. Alors que l'expression des cyclines de type D est generalement faible dans le coeur adulte, leur niveau d'expression est augmente lors de l'hypertrophie cardiaque mais il n'est pas couple a la division des myocytes. Ainsi, la reactivation de l'expression des cyclines de type D dans le coeur adulte a ete impliquee dans des processus pathophysiologiques par l'intermediaire de mecanismes distincts de ceux qui controlent la proliferation lors du developpement cardiaque. Les facteurs de croissance et autres agents exogenes regulent la production et l'activite des cyclines de type D chez les cardiomyocytes embryonnaires et adultes. Il pourrait s' averer important de comprendre comment les cardiomyocytes embryonnaires et adultes different sur le plan des mediateurs intracellulaires precis qui agissent en aval de ces molecules de signalisation, afin d'elaborer des strategies pour reactiver le cycle cellulaire des cardiomyocytes lors de la maladie cardiovasculaire du coeur adulte. Mots-cles : cyclines de type D, developpement de coeur, hypertrophie, cycle cellulaire, maladie cardiovasculaire. [Traduit par la Redaction], Introduction During embryogenesis, the heart transforms from a simple tubular structure to a specialized 4-chambered pump. The morphological changes associated with heart formation are the result of the tightly controlled [...]

The phenolic compounds present in cocoa seeds have been studied regarding health benefits, such as antioxidant and anti-inflammatory activities. Fibrosis is a wound healing response that occurs in almost all patients with chronic liver injury. A large number of cytokines and soluble intercellular mediators are related to changes in the behavior and phenotype of the hepatic stellate cell (HSC) that develop a fibrogenic and contractile phenotype leading to the development of fibrosis. The objective of this study was to assess the catechin effect in GRX liver cells in activities such as cell growth and inflammation. The GRX cells treatment with catechin induced a significant decrease in cell growth. This mechanism does not occur by apoptosis or even by autophagy because there were no alterations in expression of caspase 3 and PARP (apoptosis), and LC3 (autophagy). The expression of p27 and p53 proteins, regulators of the cell cycle, showed increased expression, while COX-2 and IL-6 mRNA showed a significant decrease in expression. This study shows that catechin decreases cell growth in GRX cells and, probably, this decrease does not occur by apoptosis or autophagy but through an anti- inflammatory effect and cell cycle arrest. Catechin also significantly decreased the production of TGF-β by GRX cells, showing a significant antifibrotic effect. Key words: GRX cells, catechin, fibrosis, inflammation. Les composes phenoliques presents dans les graines de cacao ont ete etudies en fonction de leurs bienfaits sur la sante telles leurs activites anti-oxydantes et anti-inflammatoires. La fibrose est une reponse a la cicatrisation qui survient chez presque tous les patients atteints de maladies chroniques du foie. Un grand nombre de cytokines et de mediateurs intercellulaires solubles sont relies a des changements de comportement et de phenotype des cellules stellaires hepatiques qui developpent un phenotype fibrogene et contractile qui mene au developpement de la fibrose. L'objectif de cette etude etait d'evaluer l'effet de la catechine sur les cellules hepatiques GRX, notamment sur la croissance et l'inflammation. Le traitement de cellules GRX avec de la catechine induisait une diminution significative de la croissance. Ce mecanisme ne survenait pas par apoptpse ou meme par autophagie car l'expression de la caspase 3 et de la PARP (apoptose), ou de la LC3 (autophagie) n'etait pas affectee. L'expression de p27 et de p53, des regulateurs du cycle cellulaire, etait augmentee, alors que l'expression de l'ARNm de COX-2 et de l'IL-6 etait significativement diminuee. Cette etude montre que la catechine diminue la croissance des cellules GRX et que, probablement, une telle diminution ne survient pas par apoptose ou par autophagie mais par l'intermediaire d'un effet anti-inflammatoire et d'un arret du cycle cellulaire. La catechine diminuait aussi significativement la production de TGF-β par les cellules GRX, montrant un effet antifibrotique important. Mots-cles : cellules GRX, catechine, fibrose, inflammation. [Traduit par la Redaction], Introduction Fibrosis is a wound healing response that occurs in almost all patients with chronic liver injury (Friedman 2008a). Although the mechanisms of acute injury activate fibrogenesis, the signs associated [...]

Prothymosin α is a mammalian nuclear protein involved in cell proliferation and differentiation. Here, we carried out the first study of the methylation status of ProTα genomic sequences in cell lines during differentiation as well as in tumoral tissues. We found that there is hypermethylation in all cell lines analyzed with a pattern that is characteristic of each cell type revealing specific genomic reorganizations. The decrease of ProTα mRNA during differentiation was not accompanied by changes in the methylation status. Remarkably, we found that there is hypomethylation in gastrointestinal tumors when compared with the peritumoral tissue. The biological implications of these findings are discussed. Key words: prothymosin a, cell differentiation, methylation, proliferation, gastrointestinal tumors. La prothymosine α est une proteine nucleaire de mammifere impliquee dans la proliferation et la differenciation des cellules. Nous avons realise ici la premiere etude de la methylation des sequences genomiques de la ProTα dans des lignees cellulaires en differenciation de meme que dans du tissu tumoral. Nous avons trouve une hypermethylation dans toutes les lignees cellulaires analysees avec un patron est caracteristique de chaque type cellulaire, revelant qu'il existe des reorganisations genomiques specifiques. La diminution de l'ARNm de ProTα lors de la differenciation n'etait pas accompagnee de changements de methylation. Fait remarquable, nous avons observe une hypomethylation dans les tumeurs gastrointestinales comparativement au tissu peritumoral. Les implications biologiques de ces conclusions sont discutees. Mots-cles : prothymosine a, differenciation cellulaire, methylation, proliferation, tumeurs gastrointestinales. [Traduit par la Redaction], Introduction Prothymosin alpha (ProTα) is a very abundant, highly acidic, mammalian nuclear protein whose expression is regulated by c-Myc, a pivotal transcription factor in the regulation of cell proliferation, and [...]

ARTICLE INFO Keywords: Cell cycle Colon-rectal cancer Triptolide Triprerygium wilfordii p21 ABSTRACT Triptolide, a diterpene triepoxide compound extracted from the traditional Chinese medicine herb Tripterygium wilfordii Hook F., is a [...]

ARTICLE INFO Keywords: Ovarian cancer Anti proliferation R osemary Carnosol Carnosic acid Rosmarinic acid Cisplatin Introduction Rosemary (Rosmarinus officinalis L, family Lamiaceae) is a popular culinary/medicinal herb. RE has been [...]

Non-small cell lung cancer (NSCLC) is a major health problem. Surgery is the only potential curative treatment, in spite of the high recurrence and mortality rates. Low molecular weight heparins (LMWH) have been suggested to have a positive impact on the outcome of various cancers, mainly attributed to their anticoagulant properties; yet a direct antineoplastic effect has not been excluded. We thought to evaluate the direct effect of the LMWH enoxaparin on the human lung adenocarcinomic epithelial cell line A549 and to determine potential antiproliferative and antimetastatic effects that could guide future trials. A549 cells were cultured with different concentrations of enoxaparin (1-30 U/mL). Cell counting was performed at 24, 48, and 72 h. Detection of c-Myc protein and CD44 protein was performed by electrophoresis and Western blotting. Statistical analysis was performed using paired Student's t tests. Cell counts were decreased with increasing concentrations and time of exposure to enoxaparin. This corresponds to decreased expression of c-Myc and CD44. In conclusion, enoxaparin displayed a direct dose and exposure duration dependent suppressor effect on A549 cell proliferation and the expression of both c-Myc and CD44 in vitro, suggesting reduced proliferative and metastatic potentials of these cells. Key words: CD44, c-Myc, low molecular weight heparins, non-small cell lung cancer, A549 cell line, pulmonary adenocarcinoma, lung cancer. Le cancer du poumon non a petites cellules (CPNPC) constitue un probleme de sante majeur. La chirurgie est le seul traitement curatif possible, malgre les hauts taux de recurrence et de mortalite. Les heparines de bas poids moleculaire (HBPM) pourraient avoir un impact sur revolution de divers cancers, en raison principalement de leurs proprietes anticoagulantes, bien qu'on ne puisse exclure un effet direct antineoplasique. Nous avons eu pour objectif d'evaluer l'effet direct de l'HBPM enoxaparine sur la lignee de cellules A549 issues d'adenocarcinomes de poumon humain et de determiner les effets antiproliferatifs et antimetastatiques qui pourraient guider les futures etudes. Nous avons cultive les cellules A549 avec differentes concentrations d'enoxaparine (1-30 U/mL), et effectue la numerotation des cellules a 24, 48 et 72 h. Nous avons detecte les proteines c-Myc et CD44 par electrophorese et transfert Western, et realise l'analyse statistique par un test t pour echantillons apparies. Les numerotations des cellules ont diminue avec l' augmentation des concentrations et du temps d' exposition a l'enoxaparine, ceci allant de pair avec la diminution de l'expression de c-Myc et CD44. Ainsi, l'enoxaparine a un effet suppresseur dependant de la dose et de la duree d'exposition sur la proliferation des cellules A549 et l'expression de c-Myc et de CD44 in vitro, ce qui autorise a penser a une diminution des potentiels proliferatif et metastatique de ces cellules. Mots-cles : CD44, c-Myc, heparines de bas poids moleculaire, cancer du poumon non a petites cellules, lignee cellulaire A549, adenocarcinome bronchique, cancer du poumon. [Traduit par la Redaction], Introduction Non-small cell lung cancer (NSCLC) is a major health problem worldwide (Alberg and Samet 2003). Surgery is the only potential curative treatment, despite the fact that most patients undergoing [...]

doi: 10.1016/j.phymed.2011.03.002 ABSTRACT Naringin is considered the main effective compound of Drynaria Rhizome, which is used commonly in the treatment of osteoporosis in traditional Chinese medicine. However, we found neoeriocitrin, [...]

Many invertebrates reproduce asexually by budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little investigated in didemnid ascidians. We here analyzed cell proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new brain develops de novo. To help define developmental stages of the thoracic bud, the distribution of acetylated tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D. listerianum as well as higher telomerase activity in bud tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially intense in the adult esophagus and epicardial structures that protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that form the buds. The neural complex in the thoracic bud forms from a hollow tube that appears to separate into the neural gland and the cerebral ganglion. Whereas most of the bud undergoes proliferation, including the hollow tube and the neural gland, the cerebral ganglion shows little or no proliferation. Pulse-chase labeling experiments indicate that the ganglion, as well as the myocardium, in adult zooids are instead composed of postmitotic cells., Introduction Regeneration involves resumption of morphogenesis in the adult body. Regeneration and repair of the nervous system is limited in vertebrates. However, in tunicates, which are close relatives of vertebrates [...]

We showed previously that vascular smooth muscle cells (VMSC) from spontaneously hypertensive rats (SHR) exhibit increased proliferation. The present study was undertaken to examine whether the enhanced levels of endogenous angiotensin (ANG) II and endothelin (ET)-1 contribute to the enhanced proliferation of VSMC from SHR and to further investigate the underlying mechanisms responsible for this response. The enhanced proliferation of VSMC from SHR compared with Wistar-Kyoto (WKY) rats was attenuated by losartan, BQ-123, BQ-788, and AG-1478, inhibitors of [AT.sub.1], [ET.sub.A], [ET.sub.B] and epidermal growth factor (EGF-R) receptors, respectively. In addition, BQ-123 and BQ-788 also attenuated the enhanced production of superoxide anion ([O.sup.-.sub.2]) and NADPH oxidase activity. Furthermore, diphenyleneiodonium (DPI, inhibitor of NADPH oxidase), N-acetyl-L-cysteine (NAC, [O.sup.-.sub.2] scavenger), and PP2 (inhibitor of c-Src) also inhibited the augmented proliferation of VSMC from SHR to WKY levels. In addition, the enhanced phosphorylation of EGF-R in VSMC from SHR compared with WKY was also attenuated by inhibitors of [AT.sub.1], [ET.sub.A], [ET.sub.B], and EGF-R but not by inhibitors of platelet-derived growth factor receptor or insulin-like growth factor receptor. Furthermore, the enhanced phosphorylation of ERK1/2 in VSMC from SHR was also attenuated by [AT.sub.1], [ET.sub.A], [ET.sub.B], c-Src, and EGF-R inhibitors. The phosphorylation of c-Src was significantly augmented in VSMC from SHR compared with VSMC from WKY and was attenuated by DPI and NAC. These data suggest that endogenous vasoactive peptides, through increased oxidative stress and resultant activation of c-Src, transactivate EGF-R, which through mitogen-activated protein (MAP) kinase signaling may contribute to the hyperproliferation of VSMC from SHR. oxidative stress; extracellular signal-regulated kinase 1/2; vascular smooth muscle cell proliferation; spontaneously hypertensive rat doi: 10.1152/ajpheart.00526.2010.

Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 ([InlB.sub.36-321]) was demonstrated to bind to and partially activate the HGF receptor Met. [InlB.sub.36-321] has a stable [beta]-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned [InlB.sub.36-321] (1x[InlB.sub.36-321]) and engineered a head-to-tail repeat of [InlB.sub.36-321] with a linker peptide (2x[InlB.sub.36-321]); 1x[InlB.sub.36-321] and 2x[InlB.sub.36-321] were purified from E. coli. Both 1x and 2x[InlB.sub.36-321] activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2x[InlB.sub.36-321] activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1x[InlB.sub.36-321] activated only STAT3 and ERK1/2. The 2x[InlB.sub.36-321] promoted improved motility compared with 1x[InlB.sub.36-321] and additionally stimulated proliferation equivalent to full-length HGF. Both the 1x and 2x[InlB.sub.36-321] prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2x[InlB.sub.36-321] to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair. apoptosis, angiotensin II; Akt; ERK1/2 doi: 10.1152/ajplung.00094.2010.

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phospho-ERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression. mitogen-activated protein kinase phosphatase-1; extracellular signal-regulated kinase 1/2; dual-specificity phosphatases; pregnancy; lactation doi: 10.1152/ajpendo.00341.2010.

The physiological mechanisms that preserve pancreatic [beta]-cell mass (BCM) are not fully understood. Although the regulation of islet function by the autonomic nervous system (ANS) is well established, its potential roles in BCM homeostasis and compensatory growth have not been adequately explored. The parasympathetic vagal branch of the ANS serves to facilitate gastrointestinal function, metabolism, and pancreatic islet regulation of glucose homeostasis, including insulin secretion. Given the functional importance of the vagus nerve and its branches to the liver, gut, and pancreas in control of digestion, motility, feeding behavior, and glucose metabolism, it may also play a role in BCM regulation. We have begun to examine the potential roles of the parasympathetic nervous system in short-term BCM maintenance by performing a selective bilateral celiac branch-vagus nerve transection (CVX) in normal Sprague-Dawley rats. CVX resuited in no detectable effects on basic metabolic parameters or food intake through 1 wk postsurgery. Although there were no differences in BCM or apoptosis in this 1-wk time frame, [beta]-cell proliferation was reduced 50% in the CVX rats, correlating with a marked reduction in activated protein kinase B/Akt. Unexpectedly, acinar proliferation was increased 50% in these rats. These data suggest that the ANS, via the vagus nerve, contributes to the regulation of BCM maintenance at the level of cell proliferation and may also mediate the drive for enhanced growth under physiological conditions when insulin requirements have increased. Furthermore, the disparate effects of CVX on [beta]-cell and acinar cells suggest that the endocrine and exocrine pancreas respond to different neural signals in regard to mass homeostasis. [beta]-cell mass; celiac vagotomy doi: 10.1152/ajpendo.00202.2010.

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [[sup.3]H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB ([greater than or equal to] 0.2 nM) substantially more effective than IGF-1. Surprisingly, CSMC lacked expression of PDGF receptor-[beta] (PDGF-R[beta]) upon isolation but by 4 days in vitro, CSMC gained expression of PDGF-R[beta] as shown by quantitative PCR, Western blot analysis, and immunocytochemistry; these CSMC responded to PDGF-BB but not IGF-1. PDGF-BB caused PDGF-R[beta] phosphorylation and mobilization from the surface membrane, leading to activation of both Akt and ERK signaling pathways, which were essential for subsequent proliferation. In contrast, PDGF-AA, FGF, EGF, and IGF-1 were ineffective. In vivo, control CSMC lacked expression of PDGF-R[beta]. However, this changed rapidly with TNBS-colitis, and by day 2 when CSMC proliferation in vivo is maximal, freshly isolated CSMC showed on-going PDGF-R[beta] phosphorylation that was further increased by exogenous PDGF-BB. This suggests that the onset of PDGF-R[beta] expression is a key factor in CSMC growth in vitro and in vivo, where inflammation may damage intrinsic inhibitory mechanisms and thus lead to hyperplasia. smooth muscle cell; intestine; platelet-derived growth factor; rat; mitosis; insulin-like growth factor-1; tissue culture doi: 10.1152/ajpcell.00086.2010.

Identification of renal cell progenitors and recognition of the events contributing to cell regeneration following ischemia-reperfusion injury (IRI) are a major challenge. In a mouse model of unilateral renal IRI, we demonstrated that the first cells to proliferate within injured kidneys were urothelial cells expressing the progenitor cell marker cytokeratin 14. A systematic cutting of the injured kidney revealed that these urothelial cells were located in the deep cortex at the corticomedullary junction in the vicinity of lobar vessels. Contrary to multilayered bladder urothelium, these intrarenal urothelial cells located in the upper part of the medulla constitute a monolayered barrier and express among uroplakins only uroplakin III. However, like bladder progenitors, intrarenal urothelial cells proliferated through a FGF receptor-2 (FGFR2)-mediated process. They strongly expressed FGFR2 and proliferated in vivo after recombinant FGF7 administration to control mice. In addition, IRI led to FGFR phosphorylation together with the selective upregulation of FGF7 and FGF2. Conversely, by day 2 following IRI, renal urothelial cell proliferation was significantly inhibited by FGFR2 antisense oligonucleotide administration into an intrarenal urinary space. Of notice, no significant migration of these early dividing urothelial cells was detected in the cortex within 7 days following IRI. Thus our data show that following IRI, proliferation of urothelial cells is mediated by the FGFR2 pathway and precedes tubular cell proliferation, indicating a particular sensitivity of this structure to changes caused by the ischemic process. FGFR2; ischemia doi: 10.1152/ajprenal.00585.2009.

The opioid growth factor (OGF; [Met.sup.5]-enkephalin), a constitutively expressed and tonically active inhibitory peptide, interacts with the OGF receptor (OGFr) to form an endogenous growth-regulating pathway in homeostasis. Amplification of OGF-OGFr interfacing in animal and clinical studies depresses development, neoplasia, angiogenesis, and immunity. Disruption of the OGF-OGFr axis accelerates cell proliferation and has been particularly important in wound repair. To investigate how OGF enters cells, OGF was labeled with 5,6-tetramethylrhodamine OGF (RhoOGF) to study its uptake in live cells. African green monkey kidney cells (COS-7) incubated with RhoOGF exhibited a temperature-dependent course of entry, being internalized at 37[degrees]C but not at 4[degrees]C. RhoOGF was detected in the cytoplasm 15 min after initial exposure, observed in both cytoplasm and nucleus within 30 min, and remained in the cells for as long as 5 h. A 100-fold excess of OGF or the opioid antagonist naltrexone, but not other opioid ligands (some selective for classic opioid receptors), markedly reduced entry of RhoOGF into cells. RhoOGF was functional because DNA synthesis in cells incubated with RhoOGF ([10.sup.-5] to [10.sup.-8] M) was decreased 24-36%, and was comparable to cells treated with unlabeled OGF (reductions of 26-39%). OGF internalization was dependent on clathrin-mediated endocytosis, with addition of clathrin siRNA diminishing the uptake of RhoOGF and upregulating DNA synthesis. RhoOGF clathrin-mediated endocytosis was unrelated to endosomal or Golgi pathways. Taken together, these results suggest that OGF enters cells by active transport in a saturable manner that requires clathrin-mediated endocytosis. human mesenchymal stem cells; active transport; DNA synthase; nucleocytoplasmic transport; siRNA doi: 10.1152/ajpregu.00318.2010.

We previously reported that p38 mitogen-activated protein kinase (p38) and phosphorylated ERK are upregulated in cyst epithelium of human renal dysplasia and obstructive uropathy in fetal lambs (Omori S, Fukuzawa R, Hida M, Awazu M. Kidney Int 61: 899-906, 2002; Omori S, Kitagawa H, Koike J, Fujita H, Hida M, Pringle KC, Awazu M. Kidney Int 73: 1031-1037, 2008). Dysplastic epithelium is characterized by proliferation, apoptosis, and upregulation of Pax2 and transforming growth factor (TGF)-[beta]1. In the present study, we investigated whether cyclic mechanical stretching of ureteric bud cells, a mimic of the hydrodynamic derangement after fetal urinary tract obstruction, reproduces events seen in vivo. Cyclic stretch activated p38 and ERK and upregulated Pax2 expression in a time-dependent manner in ureteric bud cells. Stretch-stimulated Pax2 expression was suppressed by a p38 inhibitor, SB203580, or a MEK inhibitor, PD98059.5-Deoxyuridine incorporation was increased by stretch at 24 h, which was also abolished by SB203580 or PD98059. On the other hand, apoptosis was not induced at 24 h by stretch but was significantly increased at 48 h. TGF-[beta]1 secretion was increased by stretch at 24 h, which was inhibited by SB203580 or PD98059. Inhibition of p38 or ERK as well as anti-TGF-[beta] antibody abolished the stretch-induced apoptosis. Finally, exogenous TGF-[beta]1 induced apoptosis of ureteric bud cells, which was inhibited by SB203580 and PD98059. In conclusion, cyclic stretch induces Pax2 upregulation, proliferation, and TGF-[beta]1-mediated apoptosis, features characteristic of dysplastic epithelium, via p38 and ERK in ureteric bud cells. dysplastic kidney; obstructive uropathy; kidney development; cell signaling doi: 10.1152/ajprenal.00402.2009.

Matrix metalloproteinases (MMPs) have been implicated in lung cyst formation in lymphangioleiomyomatosis (LAM). As doxycycline inhibits MMP activity in vivo, some patients take doxycycline, as one report has suggested a possible benefit in LAM. However, there have been no randomized controlled clinical trials of doxycycline for LAM, and any mechanism of action is unclear. Here, we examine previously proposed mechanisms of actions. Cell proliferation and adhesion were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and Cytomatrix cell adhesion kits. Apoptosis was examined by TdT-mediated dUTP nick end labeling (TUNEL) assay. MMP-2 expression was examined by quantitative real-time PCR and zymography in doxycycline-treated ELT3 cells and tumor growth using angiomyolipoma-derived tumor xenografts in nude mice. In ELT3 cells, [greater than or equal to] 25 [micro]g/ml doxycycline decreased proliferation, increased apoptosis, and caused a change in cell morphology associated with redistribution of actin stress filaments. Reduction in proliferation was also seen in human angiomyolipoma-derived cells. Cell adhesion to ECM proteins was decreased by doxycycline at 50 [micro]g/ml and prevented detachment of already adherent ceils. There was no effect of doxycycline on MMP-2 expression or activity in vitro. In the xenograft model, doxycycline (30 mg x [kg.sup.-1] x [day.sup.-1]) had no effect on tumor growth, final tumor weight, or tumor lysate MMP levels. Doxycycline at doses [greater than or equal to] 25 [micro]g/ml inhibited cell proliferation and adhesion, possibly by a toxic effect. Doxycycline had no effect on MMP-2 expression or activity or tumor growth in the xenograft model. Any possible in vivo effect is unlikely to be mediated by MMP-2 or reduced cell proliferation. lymphangioleiomyomatosis; matrix metalloproteinases doi: 10.1152/ajplung.00437.2009.

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of [K.sup.+], [Na.sup.+], and [Cl.sup.-] ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in [G.sub.0], [G.sub.1], and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of [Na.sup.+] and [Cl.sup.-] were reduced in the [G.sub.0]-[G.sub.1] phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after [Na.sup.+] and [Cl.sup.-] substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of [Na.sup.+] was observed to upregulate the activity of the [Na.sup.+]/[H.sup.+] exchanger NHE1 as well as of [Na.sup.+]-independent acid extrusion mechanisms, facilitating intracellular pH ([pH.sub.i]) recovery after acid loading and increasing [pH.sub.i]. Results using the potential sensitive dye [DiBaC.sub.4](3) showed a reduced [Cl.sup.-] conductance in S compared with [G.sub.1] followed by transmembrane potential ([E.sub.m]) hyperpolarization in S. [Cl.sup.-] substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with [G.sub.1]. Our results suggest that [Na.sup.+] regulates ELA cell proliferation by regulating intracellular pH while [Cl.sup.-] may regulate proliferation by fine-tuning of [E.sub.m] in S phase and altered [Ca.sup.2+] signaling. cell cycle; chloride channel; volume regulatory anion channel; [Na.sup.+]/[H.sup.+] exchanger 1 doi: 10.1152/ajpcell.00445.2009.

JNK is a member of the MAPK family and has essential roles in inflammation and cell differentiation and apoptosis. In recent years, there have been accumulating data indicating a novel role for JNK in cell growth and migration. In this report, we demonstrate that JNK activity is necessary for serotonin (5-HT)-induced proliferation and migration of bovine pulmonary artery smooth muscle cells (PASMCs). Stimulation with 5-HT was found to lead to activation of JNK with a maximal activation at 10 min. Inhibition of JNK with its specific inhibitor, SP-600125, or its dominant-negative form, DN-JNK, significantly reduced 5-HT-stimulated [[sup.3]H]thymidine incorporation and cyclin D1 expression. A similar inhibitory effect on SMC migration produced by 5-HT, as detected by a wound healing assay, was observed with inhibition of JNK. Furthermore, inhibition of 5-HT receptors 1B and 2A, but not inhibition of the 5-HT transporter, blocked 5-HT-induced JNK activation. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 and wortmannin had little or no effect on 5-HT-induced JNK phosphorylation, but JNK inhibitor SP-600125 and DN-JNK blocked 5-HT-stimulated phosphorylation of Akt and its downstream effectors, p70S6K1 and S6, indicating that Akt is a downstream effector of JNK. Activation of Akt by 5-HT was blocked only minimally, if at all, by inhibitors of ERK and p38 MAPK, indicating a uniqueness of JNK MAPK in this activation of Akt. Coimmunoprecipitation showed binding of Akt to JNK, further supporting the interaction of JNK and Akt. Thus JNK is a critical molecule in 5-HT-induced PASMC proliferation and migration and may act at an important point for cross talk of the MAPK and PI3K pathways. Its activation by 5-HT is initiated through 5-HT [sub.1B] and [sub.2A] receptors, and its stimulation of SMC proliferation and migration occurs through the Akt pathway. cell migration; serotonin signaling doi: 10.1152/ajplung.00281.2009.

Total parenteral nutrition (TPN) leads to a decline in phosphatidylinositol 3-kinase (PI3K)/phospho-Akt (p-Akt) activity, affecting downstream signaling, reducing epithelial cell (EC) proliferation, and contributing to intestinal mucosal atrophy. We hypothesized that promoting Akt activity would prevent these changes. We used a novel Akt-activating peptide, TCL1 (a head-to-tail dimer of the Akt-binding domain of T-cell lymphoma-1), or an inactive mutant sequence TCL1G conjugated to a transactivator of transcription peptide sequence to promote intracellular uptake. Four groups of mice were studied, enteral nutrition group (control), control mice given a functioning TCL1 (control + TCL1), TPN mice given TCL1G (control peptide, TPN + TCL1G); and TPN mice given TCL1. TPN mice given TCL1G showed a significant decrease in jejunal EC p-Akt (Ser473 and Thr308) abundance, whereas TPN + TCL1 mice showed increased p-Akt (Ser473) abundance. Phosphorylation of [beta]-catenin and glycogen synthase kinase-3[beta] (downstream targets of Akt signaling) were also decreased in the TPN + TCL1G group and completely prevented in the TPN + TCL1 group. Use of TCL1 nearly completely prevented the decline in EC proliferation seen in the TPN + TCL1G group, as well as partly returned EC apoptosis levels close to controls. The mammalian target of rapamycin pathway demonstrated a similar reduction in activity in the TPN + TCL1G group that was significantly prevented in the TPN + TCL1 group. These results support a significant loss of PI3K/p-Akt signaling upon replacing enteral nutrition with TPN, and prevention of this loss demonstrates the key importance of PI3K/p-Akt signaling in maintaining gut integrity including EC proliferation and reduction in apoptosis. intestinal epithelial cell; proliferation; apoptosis; mammalian target of rapamycin

Voltage-gated [Ca.sup.2+] channels ([Ca.sub.V]) are ubiquitously expressed in various cell types and play vital roles in regulation of cellular functions including proliferation. However, the molecular identities and function of [Ca.sub.V] remained unexplored in preadipocytes. Therefore, whole cell voltage-clamp technique, conventional/ quantitative real-time RT-PCR, Western blot, small interfering RNA (siRNA) experiments, and immunohistochemical analysis were applied in mouse primary cultured preadipocytes as well as mouse 3T3-L1 preadipocytes. The effects of [Ca.sub.V] blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold [Ca.sub.V], which could be inhibited by mibefradil, [Ni.sup.2+] ([IC.sub.50] of 200 [micro]M), and NNC55-0396. Dominant expression of [[alpha].sub.1G] mRNA was detected among [Ca.sub.V] transcripts ([[alpha].sub.1A]-[[alpha].sub.1I]), supported by expression of [Ca.sub.V]3.1 protein encoded by [[alpha].sub.1G] gene, with immunohistochemical studies and Western blot analysis, siRNA targeted for [[alpha].sub.1G] markedly inhibited [Ca.sub.V]. Dominant expression of [[alpha].sub.1G] mRNA and expression of [Ca.sub.V]3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of [[alpha].sub.1G] mRNA and [Ca.sub.V]3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type [Ca.sub.V] blocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for am also prevented cell cycle entry/progression. The present study demonstrates that the [Ca.sub.V]3.1 T-type [Ca.sup.2+] channel encoded by [[alpha].sub.1G] subtype is the dominant [Ca.sub.V] in mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development. 3T3-L1; adipose; [[alpha].sub.1G]; cell cycle doi: 10.1152/ajpcell.00488.2009.