RESUMO
Three homologues of TGF-ß exist in mammals as follows: TGF-ß1, TGF-ß2, and TGF-ß3. All three proteins share high homology in their amino acid sequence, yet each TGF-ß isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-ß proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-ß knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-ß1 ligand with a sequence from TGF-ß3 using targeted recombination to create chimeric TGF-ß1/3 knock-in mice (TGF-ß1(Lß3/Lß3)). In the TGF-ß1(Lß3/Lß3) mouse, localization and activation still occur through the TGF-ß1 latent associated peptide, but cell signaling is triggered through the TGF-ß3 ligand that binds to TGF-ß receptors. Unlike TGF-ß1(-/-) mice, the TGF-ß1(Lß3/Lß3) mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-ß3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-ß1 deficiency. However, the TGF-ß1(Lß3/Lß3) mice have a shortened life span and display tooth and bone defects, indicating that the TGF-ß homologues are not completely interchangeable. Remarkably, the TGF-ß1(Lß3/Lß3) mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-ß isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-ß pathway in human disease.
Assuntos
Glucose/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Animais , Células COS , Chlorocebus aethiops , Técnicas de Introdução de Genes , Glucose/genética , Células Hep G2 , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neovascularização Fisiológica/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Suínos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta3/genéticaRESUMO
Retinal pigment epithelium (RPE) and transforming growth factor-beta (TGF-beta) have been shown to be involved in various retinal diseases. We have studied the role of inflammatory cytokines on the expression and secretion of TGF-beta in human RPE cells (HRPE). Confluent cultures of HRPE derived from donor eyes were used. RT-PCR analyses showed that TNF-alpha and IL-1beta increased the mRNA levels of both TGF-beta1 and TGF-beta2. IFN-gamma enhanced constitutively expressed, as well as, TNF-alpha-and IL-1beta-induced TGF-beta1 mRNA levels but decreased TGF-beta2 mRNA. The effects of these cytokines on TGF-beta1 and TGF-beta2 secretion correlated with the mRNA levels. TGF-beta1 was always produced as the latent form while 21-31% of TGF-beta2 was in the active form. IFN-gamma reduced the production of active form of TGF-beta2 to 4-9%. TGF-beta3 secretion was not detectable under any of the conditions. The Real-Time PCR analysis of TGF-beta mRNAs confirmed the observed results. The TGF-beta1 and TGF-beta2 secretion was induced by TGF-beta2 and TGF-beta1, respectively. Under these conditions, the contrasting effects of IFN-gamma on TGF-beta1 and TGF-beta2 secretion were also observed. JAK inhibitor selectively inhibited IFN-gamma induced TGF-beta1 secretion and mRNA levels while reversing the inhibitory effects of IFN-gamma on TGF-beta2. Analyses of transcription factor activity strongly indicated the role of STAT-1 but not NFkappaB, C-Myc, C-Jun, SP-1, MEF-2. Our data demonstrate that IFN-gamma differentially regulates constitutively expressed, as well as, cytokine-induced TGF-beta1 and TGF-beta2 mRNA levels and secretion of TGF-betas by HRPE.