As we have found in the present study, ERK1/2 was phosphorylated in a rapid and transient manner in the process of chondrogenic differentiation induced by TGF1, but was very unstable over the late period of chondrogenesis. opposing functions in mediating transcription of cartilagespecific genes for Col2 and aggrecan. TGF1stimulated NKSF gene expression of chondrogenic regulators, Sox9, Runx2 and Ihh, was also affected by activity of PD98059 and SB203580, to different degrees. However, influences of MAPK inhibitors on gene expression were relatively minor when not treated with TGF1. Additionally, gene transcription of Smad2/3 was significantly upregulated by TGF1, but was regulated more subtly by treatment with MAPK inhibitors. Conclusions:MAPK subtypes seemed to regulate chondrogenesis with a delicate balance, interacting with the TGF1/Smads signalling pathway. == Introduction == Bone marrow mesenchymal stem cells (BMSCs) are a populace of multipotential cells capable of differentiating into mesenchymelineage cell types bothin vivoandin vitro(1). Chondrogenic potential of BMSCs has long been established and is highly encouraging for cartilage tissue repair. Differentiation begins at mesenchymal condensation and GW 501516 cells exhibit a life cycle of proliferation, differentiation, hypertrophy and apoptosis (2,3); each stage is usually characterized by appearance of specific markers. For instance, proliferative and prehypertrophic chondrocytes are characterized by expression of transcription factor Sox9 (SRYrelated gene 9), and cartilage extracellular matrix proteins such as collagen II (ColII) and aggrecan (4). Similarly, BMP6, type X collagen (ColX), alkaline phosphatase (ALP) and transcription factor Runx2 (Runtrelated transcription factor 2) are accepted as markers of chondrocyte maturation (2,5,6,7) . Chondrogenic inductionin vitrostands as a special culture system achieved by forcing aggregation of mesenchymal cells or chondroprogenitor cells to generate a micromass or pellet culture (8) and treating this with transforming growth factor (TGF) superfamily users (9,10). TGF has been widely proven to regulate cell differentiation by triggering a large variety of signalling cascades, and is tightly controlled by opinions mechanisms at different levels. Mitogenactivated protein kinases (MAPKs) have been reported as most important signalling pathway protein kinases, implicated in TGFmediated chondrocyte functions including cell proliferation, differentiation, apoptosis and inflammatory responses (4). MAPK family transduction entails a multistep kinase cascade including MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK) (11) with extracellular signalregulated protein kinase (ERK), p38 kinase and cJun Nterminal kinase (JNK). JNK has been reported to play only a very minor role in chondrogenesis as JNK phosphorylation is not affected during the process (4,12). In contrast, ERK and p38 have been proven to occupy central positions in mediating chondrocyte proliferation and related gene expression (13). However, the exact effects of ERK and p38 in chondrogenesis are still under some dispute in the current literature. ChunDo Ohet al.have reported that ERK1/2 play a negative role, whereas p38 plays a positive role in chondrogenesis of limb bud mesenchyme initiated in micromass culture (14). This view has been substantiated in studies of micromass cultures of embryonic chick limb mesenchyme cells in the absence of transforming growth factor 1 (TGF1;15) and in pellet cultures of human trabecular bonederived cells in the presence GW 501516 of TGF1 (16). In further studies, however, ERK1/2 has been reported as a positive regulator of TGF1induced aggrecan and ColII expression in adult mesenchymal progenitor cells (MPCs) (10) and chondrogenic ATDC5 cells (3). A recent statement by Bobock and Kulyk shows that MEK/ERK signalling plays diverse functions in chondrogenic differentiation of facial mesenchymal cells isolated from numerous facial primordia of chick embryo (17). These data all concern varied cell types, culture systems, GW 501516 developmental stages and culture occasions. In most studies, however, chondrogenic differentiation was observed in several days or less, and only two or three cartilagespecific markers were revealed. Thus the mechanism of MEK/ERK and p38 kinase functions in TGFinduced chondrogenesis is not thoroughly comprehended. By binding to receptors expressed at cell surfaces, TGF triggers a series of downstream signalling pathways, among which Smad proteins have been accepted as central GW 501516 mediators. Up to.