Zyxin-involved actin regulation is essential in the maintenance of vinculin focal adhesion and chondrocyte differentiation status

We have investigated the role of c-Fos in chondrocyte differentiation in vitro using both constitutive and inducible overexpression approaches in ATDC5 chondrogenic cells, which undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogenous c-fos expression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Interestingly, rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation. These data demonstrate that c-fos overexpression directly inhibits chondrocyte differentiation in vitro, and therefore these cell lines provide very useful tools for identifying novel c-Fos-responsive genes that regulate the differentiation and activity of chondrocytes.

Download Full-text

Early signalling events of autophagy

Essays in Biochemistry ◽

10.1042/bse0550001 ◽

2013 ◽

Vol 55 ◽

pp. 1-15 ◽

Cited By ~ 16

Author(s):

Laura E. Gallagher ◽

Edmond Y.W. Chan

Keyword(s):

Protein Kinase ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Mammalian Cells ◽

Mammalian Target Of Rapamycin ◽

Interacting Protein ◽

The Core ◽

Autophagy Gene ◽

Autophagy Induction ◽

Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

Download Full-text