Signals Controlling Cell Proliferation

The major interest of my laboratory is the control of proliferation in normal and cancer cells and the genes and gene-products whose interplay regulates proliferation and differentiation.

To understand how growth factors mediate cell proliferation and differentiation, we are studying the mechanism of action and the regulation of expression of fibroblast growth factors (FGF). FGF represents a large family of growth factors which signal through their interaction with tyrosine kinase receptors (FGFR) which also make-up a gene family. FGF signaling plays a major role in a variety of developmental processes, ranging from gastrulation to bone morphogenesis. Ectopic or excessive FGF expression can lead to oncogenesis. The main projects presently being carried out focus on the regulation of skeletal development by FGF signaling. Skeletal development is controlled by a small number of signaling molecules that regulate the commitment, proliferation and differentiation of osteogenic cells. Unregulated FGF signaling, due to FGFR activating mutations, causes a variety of dominant bone morphogenetic disorders in humans, including several forms of dwarfism and craniosynostosis syndromes, showing that FGF signaling plays an important role in bone development. Excessive FGF signaling alters bone development by affecting the dynamics of growth and differentiation of chondrocytes and osteoblasts, the two major cell types involved in bone formation. We are studying the biological response of these two cell types to FGF and the key pathways involved in this process.

1. In chondrocytes, we found that FGF signaling inhibits proliferation and increases apoptosis both in vitro and in vivo. These effects are cell type-specific since FGFs induce proliferation in most other cell types, and provide a logical explanation of why excessive FGF signaling causes dwarfism and chondrodysplastic syndromes. FGF-induced growth inhibition of chondrocytes requires STAT1, a signal transducing transcription factor, that can also associate with other cellular proteins involved in the control of proliferation, differentiation and apoptosis. STAT1 deficiency corrects the chondrodysplastic phenotype of mice overexpressing FGF2. We have also shown that FGF-induced growth arrest requires the activity of two Retinoblastoma (Rb) family members, p107 and p130, but not Rb itself. FGFs induce in chondrocytes a complex network of signaling and transcriptional events which ultimately result in growth arrest and induction of several aspects of chondrocytes hypertrophic differentiation. To identify the mechanisms which direct signal-transduction to growth-inhibitory pathways, we are investigating the precise role that these events play in the response of chondrocytes to FGF. One of the earliest distinguishing events following FGF treatment is the very rapid dephosphorylation of p107. Sustained activation of ERK 1/2 kinases and downregulation of AKT function also appear to contribute to the growth arrest. We have recently shown that p107 dephosphorylation is a critical early event in the growth-inhibitory response of these cells to FGF signaling and that the PP2A phosphatase targets p107 for dephosphorylation in FGF-treated chondrocytes. Our studies are presently focused on the mechanisms which “activate” PP2A to target p107 upon FGF treatment and the role of STAT1 in chondrocyte proliferation and differentiation.
   
   
2. Immature osteoblasts respond to FGF with increased proliferation, while differentiating cells undergo apoptosis. Sustained FGF signaling inhibits differentiation. We have examined the program of gene expression in osteoblasts expressing activated FGFR mutants and detected a significant down-regulation of WNT target gene expression. Concomitantly, we have observed a dramatic induction of expression of Sox2, a transcription factor of the HMG domain family, whose expression is a classical marker of embryonic stem cells. Sox2 is also induced by FGF treatment of normal osteoblasts and is clearly detectable in cranial osteoblasts in vivo. Wnt signaling is important to promote osteoblast function and high bone mass in humans and mice and thus inhibition of Wnt signaling is likely to be one important mechanism by which FGFs inhibit osteoblast differentiation. We have investigated the mechanisms by which FGF inhibits Wnt signaling in osteoblasts using Wnt-responsive reporter cell lines and biochemical and/or microarray analysis. Our results showed that FGF utilizes multiple mechanisms that lead to inhibition of Wnt-induced transcription in osteoblasts: 1) Sox2, that is strongly induced by FGF can bind to β-catenin and inhibits its transcriptional activity, 2) FGF inhibits the expression of Wnt receptors, 3) FGF-inhibits the expression of TCF1 and TCF4, 4) FGF inhibits directly the expression of several Wnt target genes.
   
   

We are now investigating the contribution of each of these factors to the antagonistic effects of FGF on Wnt signaling. An unexpected finding has been that Sox2, in addition to interfere with Wnt signaling, appears to be an important survival/proliferation factor for osteoblasts. We are thus investigating the hypothesis that Sox2 is also required to maintain osteoprogenitor cells in a stem cell-like state.

To better define the mechanisms by which FGF signaling controls osteoblast proliferation and differentiation we have also examined a mouse model of craniosynostosis induced by the Apert activating mutation in FGFR2, and correlated the in vivo observations with the studies of the properties of osteoblasts from these mutant mice in culture. Our studies suggest that the major determinant of FGFR2 induced craniosynostosis is the failure of osteoprogenitor cells with activated FGFR2 to respond to signals that would halt their recruitment/advancement at the sites where sutures should normally form.