Bisindolylmaleimide I Suppresses Fibroblast Growth Factor-mediated Activation of Erk MAP Kinase in Chondrocytes by Preventing Shp2 Association with the Frs2 and Gab1 Adaptor Proteins

Abstract

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human dwarfisms characterized by diminished long bone growth (1). In cartilage, FGFR3 alters chondrocyte proliferation and differentiation by up-regulation of cell cycle inhibitors and stimulation of cartilage matrix degradation (2-5). The anti-proliferative action of FGF 2 signaling in cartilage contrasts with the usual mitogenic response of cells to FGF stimulus (6), but the molecular basis of this paradox remains unclear. Recently, Erk MAP kinase was found as a candidate for FGFR3-mediated inhibition of chondrocyte proliferation and differentiation (7-10).Protein kinase C (PKC) comprises a family of serine/threonine kinases that phosphorylate the consensus motif RXX(S/T)XR (11). The PKCs are further divided into three subfamilies based on sequence similarities and modes of activation. The conventional PKCs (PKC␣, -␤I, -␤II, and -␥) are activated by phosphatidylserine, diacylglycerol, and Ca 2ϩ , the novel PKCs (PKC␦, -⑀, -, and -) require only phosphatidylserine and diacylglycerol, and the atypical PKCs (aPKC; PKC and -) respond to phosphatidylserine alone (12). The PKC phosphorylation motif is present in many proteins (13), implicating PKCs as broad specificity protein kinases. PKCs are involved in numerous signaling events including activation of the Erk MAP kinase pathway. This is evident by potent Erk activation in cells treated with phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), which activates both conventional PKCs and novel PKCs through binding of their diacylglycerol site (14). In PMA-treated cells, PKCs target the Erk module at the level of both Raf-1 and MEK, through direct activatory phosphorylation or indirectly (15-21). Apart from PMA-mediated Erk activation, PKCs appear to be crucial for long term Erk activation by growth factors, including FGFs (20,[22][23][24][25], as well as for oncogenic .FGF signaling in chondrocytes leads to long term Ras/Erk activation, which appears to account for the growth inhibitory outcome of FGF treatment (8, 9). To date, little is known about chondrocyte properties of FGF signaling permitting prolonged Erk activity, although slow down-regulation of mutated FGFR3 appears to be involved (30,31).

STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes

“…When heparin (Gibco-BRL) was used, the concentration was 1 g/ml. The FGFR3 kinase assays were carried out as described before (Krejci et al, 2007). Briefly, the kinase reactions were performed in 50 l of kinase buffer (60 mM HepesNaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 M Na 3 VO 4 , 1.2 mM DTT) supplemented with 2.5 g PEG, 10 M ATP and recombinant human STAT1 (500 ng), STAT3 (250 ng; Active Motif, Carlsbad, CA), or FRS2 (500 ng; Abnova, Taipei City, Taiwan) as a substrate.…”

“…Although several other cell models have been used to explore the mechanism of FGF signaling in a chondrocyte environment (Henderson et al, 2000;Yamanaka et al, 2003), RCS chondrocytes represent the best studied chondrocyte cell model to date. Using RCS chondrocytes, several essential features of FGF signaling in chondrocytes have been elucidated, including the mechanisms of FGF-mediated chondrocyte growth arrest, cytoskeletal alterations, loss of chondrocyte extracellular matrix, mechanisms of FGF and C-natriuretic peptide signaling crosstalk and others (Aikawa et al, 2001;Ben-Zvi et al, 2006;Rosenblatt-Rosen et al, 2002;Dailey et al, 2003;Raucci et al, 2004;Krejci et al, 2004;Krejci et al, 2005;Krejci et al, 2007;Priore et al, 2006). Since they respond to FGF treatment with potent growth arrest that is accompanied by the activation of STAT1 (Sahni et al, 1999), we used RCS chondrocytes in this study.…”

“…Wild-type or FGFR3-K650E were expressed in CHO cells, immunoprecipitated and subjected to a kinase assay (Krejci et al, 2007), using recombinant STAT1 or STAT3 as a substrate. Fig.…”

NF449 Is a Novel Inhibitor of Fibroblast Growth Factor Receptor 3 (FGFR3) Signaling Active in Chondrocytes and Multiple Myeloma Cells

“…Heparin (1 g/ml; Invitrogen) was used together with FGF2 for RCS chondrocyte treatment, and the cell cartilaginous matrix was removed by collagenase (type II; Invitrogen) treatment before the addition of NF449. Immunoprecipitations and FGFR3 kinase assays were performed as described (8). Briefly, the pRK7 vector encoding C-terminally FLAG-tagged K650E-FGFR3 was transfected into CHO cells using FuGENE6 reagent according to the manufacturer's protocol (Roche Applied Science).…”

“…3), we used the cell-free kinase assay to determine whether NF449 inhibits the kinase activity of K650E-FGFR3. As described in detail elsewhere (8), FLAGtagged full-length K650E-FGFR3 was expressed in CHO cells for 48 h, activated by brief FGF2 treatment, and purified by immunoprecipitation. Immunocomplexes were subjected to a kinase assay with STAT1 as a substrate and NF449 added directly to the kinase reaction.…”