FGF2 inhibits proliferation and alters the cartilage-like phenotype of RCS cells

Krejčı́, Pavel; Bryja, Vı́tězslav; Pachernı́k, Jiřı́; Hampl, Aleš; Pogue, Robert; Mekikian, Pertchoui B.; Wilcox, William R.

doi:10.1016/j.yexcr.2004.03.011

Cited by 72 publications

(104 citation statements)

References 43 publications

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“…Considering the usual mitogenic outcome of FGF signaling, the effect of the FGF signal in chondrocytes appears to be unique. It was recently shown that the Ras/Erk pathway is a candidate for FGF-mediated growth arrest, and in contrast to most other cell types, the FGF stimulus elicits a remarkably prolonged Erk activation in chondrocytes (8,9). This correlates with the known cellular responses to Erk, where transient activation appears to be crucial for mitogenic signaling of growth factors, but sustained activity frequently leads to growth arrest (44).…”

Section: Discussionmentioning

confidence: 99%

“…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)(8)(9)(10).…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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

Krejčı́

Masri

Salazar

et al. 2007

Journal of Biological Chemistry

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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).

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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

Krejčı́

Masri

Salazar

et al. 2007

Journal of Biological Chemistry

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“…A number of in vitro studies have suggested that p38 potentially regulates chondrocyte proliferation. However, both proliferative and antiproliferative effects have been reported based on the effects of p38 inhibitors in a number of culture systems (18,21,25,26). Therefore, examining how increased or decreased p38 activity in chondrocytes affects chondrocyte differentiation, proliferation, and the entire process of endochondral ossification in intact animals is of considerable interest.…”

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confidence: 99%

Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation

Zhang

Murakami

Coustry

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Accumulating in vitro evidence suggests that the p38 mitogenactivated protein kinase (MAPK) pathway is involved in endochondral ossification. To investigate the role of this pathway in endochondral ossification, we generated transgenic mice with expression in chondrocytes of a constitutively active mutant of MKK6, a MAPK kinase that specifically activates p38. These mice had a dwarf phenotype characterized by reduced chondrocyte proliferation, inhibition of hypertrophic chondrocyte differentiation, and a delay in the formation of primary and secondary ossification centers. Histological analysis with in situ hybridization showed reduced expression of Indian hedgehog, PTH͞PTH-related peptide receptor (PTH, parathyroid hormone), cyclin D1, and increased expression of p21 in chondrocytes. In addition, both in vivo and in transfected cells, p38 signaling increased the transcriptional activity of Sox9, a transcription factor essential for chondrocyte differentiation. In agreement with this observation, transgenic mice that express a constitutively active mutant of MKK6 in chondrocytes showed phenotypes similar to those of mice that overexpress SOX9 in chondrocytes. These observations are consistent with the notion that increased activity of Sox9 accounts at least in part for the phenotype caused by constitutive activation of MKK6 in chondrocytes. Therefore, our study provides in vivo evidence for the role of p38 in endochondral ossification and suggests that Sox9 is a likely downstream target of the p38 MAPK pathway.E ndochondral ossification, a process involving a cartilage intermediate, is responsible for the formation of most vertebrate skeletal elements. After the condensation of mesenchymal chondroprogenitor cells (1), cells differentiate into chondrocytes, which express cartilaginous matrix molecules and form cartilage that prefigures future skeletal elements. Endochondral bone growth takes place at the growth plate, where chondrocytes undergo unidirectional proliferation and then become hypertrophic chondrocytes. Hypertrophic chondrocytes eventually undergo apoptosis and are replaced by bone cells (2). This complex process of endochondral ossification is under the concerted regulation of various cytokines and growth factors, including fibroblast growth factors (FGFs), parathyroid hormone (PTH)-related peptide, Indian hedgehog (Ihh), and bone morphogenetic proteins (3-7).Several transcription factors, including Sox9, Sox5, Sox6, Osterix, and Runx2, have critical roles in endochondral ossification (8-12). In particular, we previously showed that Sox9 has an essential role at sequential steps in the chondrocyte differentiation pathway (8, 9). Indeed, Sox9 is needed for the condensation of chondrogenic mesenchymal cells; it is also required for the overt differentiation of these cells into chondrocytes, in part because Sox9 is needed for the expression of Sox5 and Sox6, which are also needed at this step. A further role for Sox9 is inhibition of the proliferation of chondrocytes and of the transition of these cells...

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“…RCS chondrocytes respond to FGFR3 activation (via exogenous FGF2 addition) with potent growth arrest, loss of the cartilage-like extracellular matrix, and marked alteration of their cellular shape (9,11,12). We have taken advantage of this response and adapted the FGF2/FGFR3-mediated growth arrest for rapid screening of chemical compounds for their activity against FGFR3 signaling (7).…”

Section: Nf449 Inhibits Fgfr3 Signaling In Chondrocytes-mentioning

confidence: 99%

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

Krejčı́

Murakami

Procházková

et al. 2010

Journal of Biological Chemistry

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The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site.FGFR3 (fibroblast growth factor receptor 3) is a transmembrane tyrosine kinase that serves as a receptor for the members of the fibroblast growth factor (FGF) 2 family and functions in many biological processes, including cell proliferation, differentiation, migration, and survival. To date, activating mutations in FGFR3 have been associated with several human disorders, such as skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas (1-4).Among the skeletal dysplasias, activating FGFR3 mutations cause achondroplasia, the most common form of human skeletal dysplasia, and thanatophoric dysplasia, the most common form of lethal skeletal dysplasia (1). Long bones of individuals suffering from FGFR3-related skeletal dysplasias show markedly shortened zones of chondrocyte proliferation and differentiation, whereas Fgfr3 knock-out mice and humans without functional FGFR3 demonstrate skeletal overgrowth, together implying the role of FGFR3 as a negative regulator of bone growth (5).Apart from cartilage, ϳ15% of patients suffering from multiple myeloma markedly up-regulate FGFR3 as a consequence of a t(4;14)(p16.3;q32) translocation, with a fraction of patients also harboring activating mutations in FGFR3, identical to those found in the skeletal dysplasias (2, 3). Ectopic expression of FGFR3 enhances multiple myeloma cell proliferation and survival, demonstrating the oncogenic potential of FGFR3 (6).Given its role in human disease, FGFR3 signaling represents an attractive target for therapy. There is no treatment available for achondroplasia at present, thus inciting the development of novel approaches to target FGFR3. The aim of this study was to identify novel inhibitors of FGFR3 signaling in cellular environments relevant to FGFR3-related disorders. We recently established a chondrocyte cell-based reporter assay suitable for identification of inhibitors of ...

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FGF2 inhibits proliferation and alters the cartilage-like phenotype of RCS cells

Cited by 72 publications

References 43 publications

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

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

Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation

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

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