Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogenactivated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor ␥2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.Human bone marrow-derived mesenchymal stem cells (hMSCs) 1 are multipotent, capable of differentiating into at least three lineages (osteogenic, chondrogenic, and adipogenic) when cultured under defined in vitro conditions (1-3). The hMSCs do not differentiate spontaneously, and their in vitro and in vivo osteogenic potential has been very well characterized by us and others (4 -6). When cultured in the presence of the synthetic glucocorticoid dexamethasone, ascorbic acid, and ␤-glycerophosphate (osteogenic supplements, OS), hMSCs differentiate to the osteogenic lineage, producing bone-like nodules with a mineralized extracellular matrix containing hydroxyapatite (4). The similar developmental phenomenon has also been described by others (7, 8) using bone marrow-derived cells. Other than the osteoinductive effect that OS has on MSCs, OS also acts as a mitogen (9). Presumably, the osteoinductive and mitogenic effects are due to dexamethasone present in OS because glucocorticoids are potent regulators of cellular growth and differentiation (10). However, the underlying molecular mechanisms of OS-induced mitogenic and osteogenic differentiation are presently unknown.In order to acquire a new cell phenotype, uncommitted hMSCs must undergo proliferative and differentiative changes, the two most fundamental biological processes in the life cycle of cells. One of the potential signal transduction pathways that might regulate the proliferation and differentiation of hMSCs is the MAP kin...

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.The MAP 1 kinase cascade is one of the principal intracellular signaling pathways linking activation of cell surface receptors to cytoplasmic and nuclear effectors. The MAP kinases (MAPKs), ERK1 and ERK2, have been shown to be essential for cellular proliferation as well as for acquisition and maintenance of a differentiated phenotype (1). One of the best studied models employed to examine how the MAPKs act to regulate cellular phenotypes is the rat pheochromocytoma cell line, PC12 cells. These cells respond to epidermal growth factor (EGF) treatment by an increase in mitotic rates (2). In contrast, nerve growth factor (NGF) stimulation of the PC12 cells results in their differentiation into a sympathetic neuron-like phenotype (3). There is compelling evidence that the longevity of MAPK activation governs whether these cells are stimulated either to proliferate or to withdraw from the cell cycle and differentiate into a neuronal phenotype. EGF and other mitogens provoke the evanescent activation of the MAPKs, whereas NGF treatment results in the sustained activation of this signaling pathway (4 -6). The question of how different growth factors elicit distinct biological outcomes through common signal transduction elements has provoked considerable interest resulting in a complex and controversial literature.The MAPK cascade transduces signals from receptor tyrosine kinases to two members of the Ras family of small Gproteins, Ras and Rap1, which then stimulates the sequential activation of Raf serine/threonine kinases (c-Raf and B-Raf), MEK, and the MAPKs (ERK1...

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21r'-dependent signal transduction pathway in PC12 cells. The linkage between p2l'as and MEKI was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Rafl; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Rafl-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent Mr of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Rafl, directly interacted with activated p2l' immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Rafl to bind to activated p2l'. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p2l' following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Rafl activity was not found to function within this pathway.Nerve growth factor (NGF) is responsible for the survival and differentiation of distinct subsets of neurons both within the central nervous system and in the periphery. The clonal rat pheochromocytoma cells, PC12, have proven to be a valuable model in which to investigate NGF action (15). These cells respond to NGF by cessation of division and then differentiate into a sympathetic-like neuronal phenotype. NGF initiates its actions through interaction with the proto-oncogene trk (trkA) (5, 25). TrkA possesses an intrinsic protein tyrosine kinase activity that is activated upon NGF binding, resulting in receptor autophosphorylation and phosphorylation of other proteins which participate in the signal transduction process. The detailed mechanisms through which NGF elicits its specific biochemical effects have been elusive; however, it is clear that the transmission of signals from the cell surface involves the activation of the G protein p2lras. This is followed by the serial activation of other protein kinases, driving a cascade of protein phosphorylation that mediates the specific biochemical events characteristic of NGF action through the phosphorylation of cytosolic proteins and nuclear transcription factors (7,10,18,66 (8,14,33,38