RESUMEN
We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of ß-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9 g kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.
Asunto(s)
Glycine max/genética , Hormona de Crecimiento Humana/biosíntesis , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes/biosíntesis , Semillas/genética , Secuencia de Aminoácidos , Antígenos de Plantas/genética , Globulinas/genética , Hormona de Crecimiento Humana/genética , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/genética , Proteínas de Almacenamiento de Semillas/genética , Semillas/metabolismo , Proteínas de Soja/genética , Glycine max/metabolismo , Vacuolas/metabolismoRESUMEN
GH deficiency is known to be clinically associated with a high incidence of nonalcoholic fatty liver disease, and this can be reversed by GH administration. Here we investigated the mechanistic basis for this phenomenon using engineered male mice lacking different signaling elements of the GH receptor, hepatic stat5a/b(-/-) mice and a mouse hepatoma line. We found deficient GH-dependent signal transducer and activator of transcription (STAT)-5 signaling correlates with steatosis, and through microarray analysis, quantitative PCR, and chromatin immunoprecipitation, identified putative targets of STAT5 signaling responsible for the steatosis seen on a normal diet. These targets were verified with liver-specific stat5a/b deletion in vivo, and in vitro we show that dominant-negative (DN) STAT5 increases lipid uptake in a mouse hepatoma line. Because loss of STAT5 signaling results in elevated STAT1 and STAT3 activity and intracellular lipid accumulation, we have used DN-STAT5a/b, DN-STAT1, constitutively active (CA)-STAT3, or addition of oleate/palmitate in the hepatoma line to assign which of these apply to individual targets in STAT5 signaling deficiency. These findings and published mouse models of steatosis enable us to propose elevated cd36, pparγ, and pgc1α/ß expression as primary instigators of the steatosis along with elevated fatty acid synthase, lipoprotein lipase, and very low-density lipoprotein receptor expression. Decreased fgf21 and insig2 expression may also contribute. In conclusion, despite normal plasma free fatty acids and minimal obesity, absent GH activation leads to steatosis because activated STAT5 prevents hepatic steatosis. These results raise the possibility of low-dose GH treatment for nonalcoholic fatty liver disease.
Asunto(s)
Hormona del Crecimiento/metabolismo , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Línea Celular , Hígado Graso , Regulación de la Expresión Génica/fisiología , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Análisis por Matrices de Proteínas , Receptores de Somatotropina/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT5/genéticaRESUMEN
Repression of E-cadherin expression by the transcription factor, Snail, is implicated in epithelial to mesenchymal transition and cancer progression. We show here that Integrin-Linked Kinase (ILK) regulates E-cadherin expression through Poly(ADP-ribose) polymerase-1 (PARP-1). ILK overexpression in Scp2 cells resulted in stimulation of Snail expression and loss of E-cadherin expression. Silencing of ILK, Akt or Snail resulted in re-expression of E-cadherin in PC3 cells. To elucidate the signaling pathway downstream of ILK, we identified candidate Snail promoter ILK Responsive Element (SIRE) binding proteins. PARP-1 was identified as a SIRE-binding protein. ILK silencing inhibited binding of PARP-1 to SIRE. PARP-1 silencing resulted in inhibition of Snail and ZEB1, leading to up-regulation of E-cadherin. We suggest a model in which ILK represses E-cadherin expression by regulating PARP-1, leading to the binding of PARP-1 to SIRE and modulation of Snail expression.