RESUMEN
Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and ß1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.
Asunto(s)
Alginatos/farmacología , Hidrogeles/farmacología , Células Madre Mesenquimatosas/metabolismo , Oligopéptidos/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/farmacología , Humanos , Integrinas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Mitógenos/farmacologíaRESUMEN
Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH (zRICH) protein were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.
Asunto(s)
Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Nervio Óptico/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Western Blotting , Células PC12 , Hidrolasas Diéster Fosfóricas , Ratas , TransfecciónRESUMEN
Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.
Asunto(s)
ADN/metabolismo , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Co-Represor 1 de Receptor Nuclear/química , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 2 de Receptor Nuclear/química , Co-Represor 2 de Receptor Nuclear/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Estradiol/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Humanos , Ligandos , Datos de Secuencia Molecular , PPAR alfa/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Elementos de Respuesta/genética , Transcripción Genética/efectos de los fármacosRESUMEN
UNLABELLED: We tested the hypothesis that the pathogenesis of alcoholic liver injury is mediated by epigenetic changes in regulatory genes that result from the induction of aberrant methionine metabolism by ethanol feeding. Five-month-old cystathionine beta synthase heterozygous and wild-type C57BL/6J littermate mice were fed liquid control or ethanol diets by intragastric infusion for 4 weeks. Both ethanol-fed groups showed typical histopathology of alcoholic steatohepatitis, with reduction in liver S-adenosylmethionine (SAM), elevation in liver S-adenosylhomocysteine (SAH), and reduction in the SAM/SAH ratio with interactions of ethanol and genotype effects. Hepatic endoplasmic reticulum stress signals including glucose-regulated protein-78 (GRP78), activating transcription factor 4, growth arrest and DNA damage-inducible gene 153 (GADD153), caspase 12, and transcription factor sterol response element binding protein-1c (SREBP-1c) were up-regulated in ethanol-fed mice with genotype interactions and negative correlations with the SAM/SAH ratio. Immunohistochemical staining showed reduction in trimethylated histone H3 lysine-9 (3meH3K9) protein levels in centrilobular regions in both ethanol groups, with no changes in trimethylated histone H3 lysine-4 levels. The chromatin immunoprecipitation assay revealed a decrease in levels of suppressor chromatin marker 3meH3K9 in the promoter regions of GRP78, SREBP-1c, and GADD153 in ethanol-treated heterozygous cystathionine beta synthase mice. The messenger RNA expression of the histone H3K9 methyltransferase EHMT2 (G9a) was selectively decreased in ethanol-fed mice. CONCLUSION: The pathogenesis of alcoholic steatohepatitis is mediated in part through the effects of altered methionine metabolism on epigenetic regulation of pathways of endoplasmic reticulum stress relating to apoptosis and lipogenesis.