QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 October 2005
doi: 10.1242/jcs.02618

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Krejci, P. Articles by Wilcox, W. R. Search for Related Content PubMed PubMed Citation Articles by Krejci, P. Articles by Wilcox, W. R. Social Bookmarking                         What's this?

Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis

¹ Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
² INSERM U589, Institut Louis Bugnard, 31403 Toulouse, France
³ Orthopaedic Research Laboratories, University of Washington, Seattle, WA 98195, USA
⁴ Department of Pediatrics, UCLA School of Medicine, CA 90095, USA

View larger version (40K): [in a new window]   Fig. 1. CNP antagonizes FGF2-mediated inhibition of RCS proliferation. (A,B) Cells were treated with FGF2 and either CNP or pCPT-cGMP for 72 hours and counted. Data represent the average from four wells with the indicated standard deviation. (B) Cells not treated with FGF2 were given an artificial value of 0.08 on logarithmic x-axis. (C) Cells were treated with FGF2 and either CNP (Experiment 1) or pCPT-cGMP (Experiment 2) for 24 hours and analyzed for DNA content by flow-cytometry. Note the FGF2-mediated accumulation of cells in the G1 phase of cell cycle that was partially reversed by CNP or pCPT-cGMP.

View larger version (25K): [in a new window]   Fig. 2. CNP inhibits FGF2-mediated activation of Erk MAP kinase. (A,B) Serum-starved cells were treated with FGF2 and either CNP or pCPT-cGMP for 30 minutes and analyzed for Erk1/2 phosphorylation by WB (A) or Erk activity by kinase assay using Elk as a substrate (B). Elk phosphorylation at Ser383 was determined by WB. Levels of total Erk1/2 serve as a loading control. (C) Cells were treated with FGF2 alone or together with CNP or pCPT-cGMP for different times up to 12 hours and analyzed for Erk phosphorylation by WB. The WB signal was quantified by densitometry and normalized to total Erk expression. The percentage of Erk phosphorylation relative to cells treated with FGF2 alone (100%) for each time point was graphed with the indicated standard deviation. Note that in short-term FGF2 treatment (1 hour), Erk activation was nearly completely inhibited by CNP or pCPT-cGMP in contrast to longer treatments (2-12 hours) where both CNP and pCPT-cGMP only lowered Erk activation by approximately 50%.

View larger version (21K): [in a new window]   Fig. 3. CNP-mediated generation of cGMP in RCS cells. (A) Cells (1x105 per well) were treated with CNP for the indicated times and the intracellular cGMP concentration was determined by ELISA. The cGMP concentration in untreated cells was 0.023 pmol/well. The data represent the average from two wells. (B) Cells (1x105 per well) were treated as indicated and the intracellular cGMP concentration was determined by ELISA. Note that the addition of fresh CNP failed to induce cGMP in cells previously treated with CNP. The data represent the average from two wells with the indicated range. (C) Cells (1x105 per well) were pretreated with PDE inhibitors 30 minutes before treatment with CNP (5 hours) and the intracellular cGMP concentration was determined by ELISA. The data represent the average from two wells with the indicated range.

View larger version (18K): [in a new window]   Fig. 4. CNP inhibits FGF2-mediated Erk MAP kinase activation via PKG. (A) Serum-starved cells were pre-treated with the PKG inhibitor KT5823 for 30 minutes before treatment with FGF2 and CNP (30 minutes) and analyzed for Erk phosphorylation by WB. Levels of total Erk2 serve as a loading control. (B) Cells were treated with FGF2 and cGMP analogs that either activate PKG (pCPT-cGMP) or inhibit PKG (Rp-8-pCPT-cGMPS; Rp-8-Br-PET-cGMPS) for 72 hours and counted. The data represent the average from four wells with the indicated standard deviation.

View larger version (33K): [in a new window]   Fig. 5. CNP inhibits FGF2-mediated activation of the Erk MAP kinase pathway at the level of Raf-1. (A) Serum-starved cells were treated with FGF2 and CNP for 30 minutes and Raf-1 activation (phosphorylation at Ser338) was determined by WB. Levels of total Raf-1 serve as a loading control. The WB signal was quantified by densitometry and graphed. (B) Serum-starved cells were treated as indicated for 30 minutes in media containing 1 µg/ml of heparin. Raf-1 was immunoprecipitated (IP) and its kinase activity was determined by kinase assay using MEK1 as a substrate. MEK1 phosphorylation at Ser217/221 was determined by WB. Phosphorylated Erk1/2 was determined in total cell lysates used for Raf-1 IP. Levels of total Raf-1, MEK1 and Erk1/2 serve as a loading controls. `–Ab', no antibody used for IP. The WB signal was quantified by densitometry and graphed. (C) Serum-starved cells were treated as indicated for 30 minutes and activated Ras (Ras-GST) was determined as described in the Materials and methods. Erk1/2 phosphorylation was detected in total cell lysates used for the Ras activity assay. Levels of total Ras and Erk2 serve as loading controls.

View larger version (27K): [in a new window]   Fig. 6. Modulation of FRS2 phosphorylation by FGF2 and CNP. (A) Serum-starved cells were treated with FGF2 and CNP for 30 minutes and immunoprecipitated FRS2 was analyzed for tyrosine phosphorylation by WB with the 4G10 antibody. The WB signal was quantified by densitometry and graphed. (B) Serum-starved cells were treated as indicated for 30 minutes and FRS2, phosphorylated Erk1/2, and total Erk1/2 were detected by WB. Note that upon FGF2 treatment, FRS2 underwent an electrophoretic mobility shift that disappeared when Erk activation was prevented by CNP or U0126. (C) Serum-starved cells were treated as indicated for 30 minutes and FRS2 was detected by WB. Note that KT5823 abolishes the FRS2 electrophoretic mobility shift seen in CNP-treated cells.

View larger version (79K): [in a new window]   Fig. 7. CNP counteracts FGF2-mediated loss of the extracellular matrix and fibronectin induction in RCS cells. (A) Cells were treated with FGF2 and CNP for 72 hours and the amount of extracellular matrix was determined by Alcian blue staining (left panel, 200x). The corresponding darkfield photograph is also shown (right panel, 200x). (B) Cells were treated with FGF2 and CNP for 72 hours and the culture media, conditioned by cells for last 24 hours of treatment, was resolved by reducing SDS-PAGE followed by Coomassie Brilliant Blue staining. Note that the volume of the sample loaded corresponds to the 1/100th of the total media conditioned by the amount of cells indicated. (C) Cells were treated with FGF2 and CNP for up to 48 hours and the amount of fibronectin mRNA was determined by real-time RT-PCR. Quantities of the transcript are relative to untreated cells at each time point.

View larger version (39K): [in a new window]   Fig. 8. CNP effects on FGF2-mediated induction and activation of matrix-remodeling molecules. (A) Cells were treated with FGF2 for 4 or 24 hours and the expression of the listed molecules was assayed by RT-PCR. Levels of Gapdh serve as a control for template quantity. (B) Cells were treated with FGF2 and either CNP or the MEK inhibitor U0126 for 4 hours and the amounts of Mmp3, Mmp10, Mmp13, Adamts1 and Adamts5 mRNAs were determined by real-time RT-PCR. Transcript quantities are relative to untreated cells. Mmp2 and Mmp9 were not studied due to the possible nonspecific actions of U0126 during the prolonged cultivation time necessary for their induction. (C,D) Cells were treated with FGF2 and CNP for 72 hours and the conditioned culture media was analyzed for MMP activity (C) by zymography using gelatin (upper figure) or casein (lower figure) as a substrate or (D) for presence of MMP13 by WB. The zymography signal was quantified by densitometry and graphed.

View larger version (23K): [in a new window]   Fig. 9. FGF2 and CNP effects on extracellular matrix homeostasis. (A,C) Cells were treated with FGF2 and CNP for 48 hours in the presence of [35S]sulfate (A) or [3H]proline (C) and the amount of incorporated radioactivity in the cell layer was determined by liquid scintillation. Note the potent regulation of [35S]sulfate incorporation by both FGF2 and CNP in contrast to [3H]proline incorporation that was only mildly enhanced by CNP. (B,D) Cells were pulsed with [35S]sulfate (B) or [3H]proline (D) for 12 hours prior to 48 or 72 hours of treatment with FGF2 and CNP. The remaining incorporated radioactivity was determined by liquid scintillation. Note that the FGF2-mediated decrease of incorporated [3H]proline required 72 hours of treatment. The data represent the average from six wells with the indicated standard deviations. (E) Cells were treated with FGF2 and CNP for 48 hours, harvested by either scraping or trypsinization and both the wet and dry mass of the pellets was determined. The data represent the average from eight wells with the indicated standard deviations. Statistically significant differences are highlighted (ANOVA; *P<0.05, **P<0.01).

View larger version (21K): [in a new window]   Fig. 10. Interaction of FGF2 and CNP signaling in RCS cells. FGF2 inhibits proliferation and triggers matrix degradation in RCS cells through activation of the Erk MAP kinase pathway. CNP signaling counteracts FGF2 effects by inhibiting the Erk pathway at the level of Raf-1. In addition, CNP directly stimulates RCS matrix production thereby further compensating for the FGF2-mediated matrix loss. The inhibitory effect of FGF2 on RCS matrix production is not presently clear (dashed line).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter