RESUMEN
OBJECTIVE: Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS: MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS: The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS: Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Osteoartritis de la Rodilla/tratamiento farmacológico , Plasma Rico en Plaquetas , Células Madre/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteína Morfogenética Ósea 4/metabolismo , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Colágeno Tipo II/biosíntesis , Femenino , Ratas , Ratas Desnudas , Trasplante de Células Madre , Células Madre/metabolismo , Rodilla de Cuadrúpedos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The POU-homeodomain transcription factor Pit-1 is required for the differentiation of the anterior pituitary cells and the expression of their hormone products. Pit-1beta, an alternate splicing isoform, has diametrically different outcomes when it is expressed in different cell types. Pit-1beta acts as a transcriptional repressor of prolactin (PRL) and growth hormone genes in pituitary cells, and as a transcriptional activator in non-pituitary cells. In order to explore these differences, we: (1) identified the transcriptional cofactors necessary for reconstitution of repression in non-pituitary cells; (2) tested the effect of the beta-domain on heterodimerization with Pit-1 and physical interaction with the co-activator CREB binding protein (CBP); and (3) determined the beta-domain sidechain chemistry requirements for repression. Co-expression of both Pit-1 isoforms reconstituted the repression of the PRL promoter in non-pituitary cells. The beta-domain allowed heterodimerization with Pit-1 but blocked physical interaction with CBP, and specific chemical properties of the beta-domain beyond hydrophobicity were dispensable. These data strongly suggest that Pit-1beta represses hormone gene expression by heterodimerizing with Pit-1 and interfering with the assembly of the Pit-1-CBP complex required for PRL promoter activity in pituitary cells.
Asunto(s)
Regulación de la Expresión Génica , Prolactina/genética , Prolactina/metabolismo , Regiones Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Factor de Transcripción Pit-1/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Dimerización , Células HeLa , Humanos , Datos de Secuencia Molecular , Hipófisis/citología , Hipófisis/metabolismo , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción Pit-1/química , Factor de Transcripción Pit-1/genéticaRESUMEN
Many transcription factors are expressed as multiple isoforms with distinct effects on the regulation of gene expression, and the functional consequences of structural differences between transcription factor isoforms may allow for precise control of gene expression. The pituitary transcription factor isoforms Pit-1 and Pit-1beta differentially regulate anterior pituitary hormone gene expression. Pit-1 is required for the development of and appropriate hormone expression by anterior pituitary somatotrophs and lactotrophs. Pit-1beta differs structurally from Pit-1 by the splice-insertion of the 26-residue beta-domain in the trans-activation domain, and it differs functionally from Pit-1 in that it represses expression of the prolactin promoter in a cell-type specific manner. In order to identify signal and promoter context requirements for repression by Pit-1beta, we examined its function in the presence of physiological regulatory signals as well as wild-type and mutant Pit-1-dependent target promoters. Here, we demonstrate that Pit-1beta impairs recruitment of cAMP response element-binding protein (CREB)-binding protein to the promoters that it represses. In addition, we show that repression of target promoter activity, reduction in promoter histone acetylation, and decrease of CREB-binding protein recruitment all depend on promoter context. These findings provide a mechanism for promoter-specific repression by Pit-1beta.