The commitment and differentiation of pluripotent mesenchymal stern cells is governed by developmental factors, cytokines and transcription factors which act in concert to determine the terminal differentiated state. They can be induced to commit to a variety of lineages such as adipocytes, osteoblasts, chondrocytes, rnyoblasts and fibroblasts. Our studies focused initially on the mechanism of action of parathyroid hormone related protein (PTHrP) in the inhibition of adipogenesis. In vitro studies that employed the pre-adipocytic cell line 3T3-Ll demonstrated the ability of PTHrP to delay the terminal differentiation of adipocytes by a cyclic AMP-protein kinase A dependent pathway that ultimately led to the phosphorylation and downregulation of the adipocyte differentiation factor Peroxisome Proliferator Activator Receptor Gamma (PPARy). This inhibitory effect of PTHrP on adipogenesis was examined further in the earlier uncommitted pluripotent mesenchymal C3H 1 OTY2 cell line. These cells can be induced to undergo adipogenesis by induction with bone morphogenetic protein 2 (BMP2). However, PTHrP treatment reduced the capacity of BMP2 to direct differentiation along the adipogenic lineage. In contrast to the inhibitory effect that PTHrP had on adipogenesis, PTHrP was observed to enhance osteogenesis. Both PTHrP effects appeared to be the result of its ability to sensitize C3H 1 OTY2 cells to the agonist BMP2. The increased efficacy of BMP2 was determined to be a result of increased bone morphogenetic protein 1 A receptor expression which was a consequence of the capacity of PTHrP to stimulate protein kinase C activity. Efforts to identify intracellular partners involved with the intracrine function of PTHrP resulted in the identification of the nuclear orphan receptor Rev ErbA as a candidate nuclear partner. Overexpression studies involving both a nuclear form of PTHrP and Rev ErbA resulted in enhanced osteoblastic differentiation ofC3H lOTYz cells. We further analyzed the regulation ofthe commitment process of cells of mesenchymal origin to the osteoblastic lineage by performing genomic scans in both the human and mouse genomes for genes harboring a Core Binding Factor alpha 1 (CBFAI) consensus binding sequence within their proximal promoter regions. Genes that contained similar response elements at complementary positions from the mRNA transcription start site were scored as potential candidate genes. These findings can, in the future, be complemented with microarray analysis to determine the expression profile of C3H 1 OTYz cells following introduction of human and mouse CBFAI. Genes that are also scored in the genomic analysis and that are activated by the mouse and human CBFAl can subsequently be examined for direct regulation by CBFAl. This therefore represents a novel approach to the search for direct gene targets of the osteoblast-regulatory factor, CBFAI and should lead to an increased understanding ofthe regulation of osteoblast development.

L'engagement et la differenciation des cellules souches pluripotentes du mesenchyme sont dependants de facteurs developpementaux, de cytokines et de facteurs de transcription qui agissent ensemble pour determiner le stade terminal de differenciation. Elles peuvent etre induites a s'engager en plusieurs lignees telles que les adipocytes, les osteoblastes, les chondrocytes, les myoblastes et les fibroblastes. Notre etude avait pour but initial le mecanisme d'action de la proteine apparentee a !'hormone parathyroide (PTHrP) dans !'inhibition de l'adipogenese. Les etudes in vitro utilisant la lignee cellulaire preadipocytaire 3T3-Ll ont demontre que les PTHrPs retardent la differenciation terminale des adipocytes via une voie de signalisation impliquant la proteine kinase A, AMP cyclique dependante, conduisant a la phosphorylation et a la regulation negative du facteur de differenciation adipocytaire Peroxisome Proliferator Activator Receptor Gamma (PPARy). L'effet inhibiteur de la PTHrP sur l'adipogenese a ete examine plus en details dans la lignee cellulaire pluripotente indifferenciee du mesenchyme C3H lOTK Ces cellules peuvent etre induite en adipogenese sous l'effet de la Bone Morphogenetic Protein 2 (BMP2). Cependant, un traitement a la PTHrP reduit la capacite de la BMP2 a amener ces cellules en differenciation vers une lignee adipocytaire. A !'inverse de !'inhibition de la PTHrP sur l'adipogenese, un effet positif sur 1' osteogenese est observe. Ces deux phenomenes de la PTHrP semblent etre le resultat de sa capacite a sensibiliser les cellules C3H lOTY:​z a l'agoniste BMP2. Cette efficacite croissante de la BMP2 est consequente de !'augmentation de !'expression du recepteur de la proteine morphogenetique de I' os lA, resultant de la capacite de la PTHrP a stimuler l'activite de la proteine kinase C. Les efforts pour identifier les partenaires intracellulaires participant a la fonction intracrine des PTHrPs a permis d'identifier le recepteur nucleaire orphelin Rev ErbA comme partenaire nucleaire potentiel. Les etudes de surexpression impliquant les formes nucleaires a la fois de PTHrP et de Rev ErbA ont montre une augmentation de la differenciation des cellules osteoblastiques C3H IOTYz. Afin d'analyser plus en details le processus d'engagement des cellules d'origine du mesenchyme en lignee osteoblastique, nous avons realise un criblage genomique des genes portant une sequence consensus de liaison en dedans de leurs regions promoteurs proximaux respectifs, pour CBFAl (Core Binding Factor alpha 1), chez l'homme et la souris. Les genes contenant des elements de reponse semblables sur les positions complementaires a partir du site d'initiation de la transcription de 1' ARN messager, ont ete repertories comme genes potentiels. Ces resultats seront ensuite completes par une analyse a biopuce d' ADN afin de determiner le profil d'expression des cellules C3H lOTYz apres introduction de CBFAl humain et murin. Les genes cribles dans !'analyse genomique ainsi que ceux qui ont ete actives par CBFAl humain et murin seront examines ulterieurement dans la regulation directe par CBF Al. Ceci represente une nouvelle approche d'identification des genes cibles modulant directement le facteur regulateur osteoblastique, CBF Al et devrait amener a une comprehension plus approfondie des mecanismes regulant le developpement osteoblastique.