Prostaglandin biosynthesis by lapine articular chondrocytes in culture

Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E₂, 6-oxoprostaglandin F_1α, (the stable metabolite of prostacyclin) and prostaglandin F_2α. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B₂ (the stable metabolite of thromboxane A₂). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [³H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E₂ relative to 6-oxo-prostaglandin F_1α than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in short-term culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prosta-glandins found in articular cartilage. Minimal quantities of thromboxane B₂ were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F_1α relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondrocytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prosta-glandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation.     

Estradiol and tamoxifen stimulation of lapine articular chondrocyte prostaglandin synthesis

The effect of estradiol and tamoxifen on prostaglandin (PG) synthesis by rabbit articular chondrocytes in secondary monolayer cultures was investigated. Radioimmunoassay for PGE₂, PGF_2α, 6-oxo-PGF_1α and thromboxane B₂ was performed on media from cultures containing estradiol and tamoxifen (10⁻¹²M-10⁻⁷-M). Radiometric thin-layer chromatography was also carried out. The time course of estradiol/tamoxifen effect on chondrocyte PG synthesis was evaluated and its relationship to cell density in culture examined. Estradiol stimulated the synthesis of PGs by chondrocytes. Stimulation was noted at picomolar concentrations of estradiol without further stimulation at markedly higher concentrations. In time studies, after a lag, the effect of estradiol was present fully by 5 hrs, remained steady for 24 hrs and then declined by 48 hrs. Estradiol stimulation of PG synthesis was dependent upon chondrocyte culture plating density. Tamoxifen stimulated chondrocyte PG synthesis to relatively lower levels than estradiol. The characteristics of estradiol/tamoxifen stimulation of chondrocyte PG synthesis suggest a mechanism involving estradiol cytoplasmic receptors.     

Effects of transforming growth factor β and interleukin-1β on [3H]thymidine incorporation by human articular chondrocytes in vitro

This is a study of the regulation of human articular chondrocyte proliferation by transforming growth factor β (TGFβ) and interleukin-1β (IL-1β) in vitro. Human articular chondrocytes were cultured at different cell densities on plastic and on a collagen substratum, in the presence and absence of serum. The effects TGFβ amd IL-1β on proliferation of chondrocytes, as determined by [³H]thymidine incorporation, under these conditions of culture were examined. TGFβ was found to have both stimulatory and inhibitory effects on chondrocytes in vitro. Interactions between TGFβ and growth factors present in serum influence the modulation of chondrocyte proliferation by TGFβ. IL-1β caused a significant reduction of the TGFβ-stimulated increase in chondrocyte proliferation. The complex inter-relationships between TGFβ and IL-1β on chondrocytes have implications for cartilage repair.     

Src kinase inhibition promotes the chondrocyte phenotype

Regulated differentiation of chondrocytes is essential for both normal skeletal development and maintenance of articular cartilage. The intracellular pathways that control these events are incompletely understood, and our ability to modulate the chondrocyte phenotype in vivo or in vitro is therefore limited. Here we examine the role played by one prominent group of intracellular signalling proteins, the Src family kinases, in regulating the chondrocyte phenotype. We show that the Src family kinase Lyn exhibits a dynamic expression pattern in the chondrogenic cell line ATDC5 and in a mixed population of embryonic mouse chondrocytes in high-density monolayer culture. Inhibition of Src kinase activity using the pharmacological compound PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine) strongly reduced the number of primary mouse chondrocytes. In parallel, PP2 treatment increased the expression of both early markers (such as Sox9, collagen type II, aggrecan and xylosyltransferases) and late markers (collagen type X, Indian hedgehog and p57) markers of chondrocyte differentiation. Interestingly, PP2 repressed the expression of the Src family members Lyn, Frk and Hck. It also reversed morphological de-differentiation of chondrocytes in monolayer culture and induced rounding of chondrocytes, and reduced stress fibre formation and focal adhesion kinase phosphorylation. We conclude that the Src kinase inhibitor PP2 promotes chondrogenic gene expression and morphology in monolayer culture. Strategies to block Src activity might therefore be useful both in tissue engineering of cartilage and in the maintenance of the chondrocyte phenotype in diseases such as osteoarthritis.     

Differential response of articular chondrocyte populations to thromboxane B2 and analogs of prostaglandin cyclic endoperoxides

The effects of two unsaturated fatty acids, prostaglandin E₂, thromboxane B₂ (TxB₂) and 2 analogs of PG endoperoxide on monolayer cultures of rabbit articular chondrocytes have been studied. Arachidonic and linoleic acids had no effect on either DNA or sulfated-glycosaminoglycan biosynthesis, while 13,14 dihydro-PGE₂ and PGE₂ markedly inhibited the former. Two epoxymethano analogs of endoperoxide PGH₂ (Em-PGH₂) at concentrations of 2.5 and 25 μg/ml stimulated cell proliferation while reducing ³⁵SO₄ incorporation. By contrast, Em-PGH₂ at lower concentrations (0.25 – 250 ng/ml) inhibited DNA synthesis in a dose-dependent manner. TxB₂ at 2.5 μg/ml did not alter cellular proliferation. At lower concentrations, 2.5 and 25 ng/ml, TxB₂ significantly stimulated sulfated-glycosaminoglycan biosynthesis in at least one of the chondrocyte populations tested. The results also demonstrated marked differences in the effects of TxB₂ and the Em-PGH₂ analogs on the partitioning of newly synthesized sulfated-proteoglycan between the cells and medium of these cell cultures.     

A conditioned medium (CM) factor produced by chondrocytes that promotes their own differentiation

This report presents evidence demonstrating that chick embryo chondrocyte cultures release into the culture medium a factor(s) which itself can act on chondrocytes to promote their own differentiation. Conditioned medium (CM) stimulates the synthesis of both sulfated mucopolysaccharides, as shown by increased incorporation of ³⁵SO₄ or glucose-¹⁴C into hyaluronidase-sensitive material, and collagen. However, protein synthesis, DNA synthesis, and cell number are not affected. While the identity of the factor is not yet known, it is nondialyzable, trypsin-and heat-sensitive. The factor is evidently a specialized product of chondrocytes, because it is not made by unexpressed chondrocytes or differentiated pigmented retina cultures. CM works rapidly on test cultures and has a significant effect on ³⁵SO₄ incorporation after 2 hr of treatment. In addition, the effect is relatively stable and is not reversed when CM is replaced with fresh medium. The results are significant in that they demonstrate that chondrocytes produce a factor that promotes their own differentiation, as defined in terms of the synthesis of two distinct specialized products.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

The effect of phorbol esters on the proliferation of C3H-10T1/2 mouse fibroblasts: Consideration of both stimulatory and inhibitory effects

TPA stimulates cell cycle activation in both serum-deprived and density-inhibited cultures. The cells reestablish cycle arrest after no more than one generation, and addition of fresh drug produces no further response. However, cells freshly trypsinized can respond with a series of repetitive generations resulting in 3.5–4.0 population doublings over 72 hrs. In kinetic pulse experiments TPA enhanced ³H-thymidine incorporation in densityinhibited cells stimulated by fresh serum but only after markedly suppressing incorporation 8–13 hrs after serum stimulation. When cells arrested by serum deprivation were pretreated with TPA, fresh serum stimulation led to initiation of ³H-TdR incorporation 5 hrs earlier than untreated controls. However, TPA addition at the time of serum stimulation did not lead to a suppression at 8–13 hrs, whereas enhancement was observed during peak incorporation times regardless of whether the cells were pretreated with TPA during serum deprivation. The results support the concept that there can exist within G₁ multiple states of responsiveness to phorbol esters. These pharmacologically induced states may be correlated with corresponding physiological states of the G₁ phase of cell cycle.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

Sex-dependent effects of 17-beta-estradiol on chondrocyte differentiation in culture

This study examined the effects of 17-beta-estradiol (E₂) on chondrocyte differentiation in vitro. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used to determine whether the effects of E₂ were dependent on the stage of chondrocyte maturation and whether they were sex-specific. [³H]-incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators of differentiation. Alakaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. Specificity of the E₂ effects was assessed using 17-alpha-estradiol. The role of fetal bovine serum and phenol red in the culture medium was also addressed. The results demonstrated that E₂ decreases cell number and [³H]-incorporation in female chondrocytes, indicating that it promotes differentiation of these cells. Alkaline phosphatase specific activity is stimulated in both growth zone and resting zone cells, but the effect is greater in the less mature resting zone chondrocytes. The increase in enzyme activity is targeted to the matrix vesicles in both cell types, but the fold increase is greater in the growth zone cells. In male chondrocytes, there was a decrease in [³H]-incorporation at high E₂ concentrations in resting zone cells at the earliest time point examined (12 hours) and a slight stimulation in alkaline phosphatase activity in growth zone cells at 24 hours. Cells cultured in serum-free medium exhibited a dose-dependent inhibition in alkaline phosphatase activity when cultured with E₂, even in the presence of phenol red. E₂-stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. E₂ increased percent collagen production in female cells only; the magnitude of the effect was greatest in the resting zone chondrocyte cultures. The results of this study indicate that the effects of E₂ are dependent on time of exposure, presence of serum, and the sex and state of maturation of the chondrocytes. E₂-stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles. © 1993 Wiley-Liss, Inc.     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

The core protein of growth plate perlecan binds FGF-18 and alters its mitogenic effect on chondrocytes

Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth plate chondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a K_d of 145 nM. Near saturation, ∼103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a K_d of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full-length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated ³H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.     

Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Objective

To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system.

Methods

Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using ³⁵SO₄²⁻-labelling and the dimethylmethylene blue assay. ³H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured.

Results

The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days. ³H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the ³⁵SO₄²⁻ incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts.

Conclusion

The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

The in vitro production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism $\text{in vivo}$.     

The production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism .     

Vasopressin stimulates sulfate incorporation into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture

The effects of lysine vasopressin (1–100 ng/ml) on the 24 h incorporation of [³⁵SO₄⁻] into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture has been investigated. The hormone enhances sulfate incorporation into proteoglycans released in the medium and those associated with the cell layer. This enhancement was independent of cell density or stimulation of cell division by the hormone or calf serum. These observations provide evidence that the hormone stimulation of sulfate incorporation is not directly linked to hormone stimulation of cell division.     

Human chondrocytes in tridimensional culture

Summary Cartilage was taken from the macroscopically normal part of human femoral heads immediately after orthopedic surgical operations for total prothesis consecuitive to hip arthrosis. After clostridial collagenase digestion and repeated washings, chondrocytes (10⁶ cells) were cultivated in a gyrotory shaker (100 rpm). Under these conditions, cells were kept in suspension and after 3 to 5 d formed a flaky aggregate which, on Day 10, became dense. These chondrocytes were morphologically differentiated: they had a round shape, were situated inside cavities, and were surrounded by a new matrix. Histochemical methods showed the presence of collagen and polysaccharides in cell cytoplasm and in intercellular matrix, and the immunofluorescence method using specific antisera (anticartilage proteoglycans and anti-type II collagen) showed that these two constituts were in tentercellular matrix. The measurement of the amounts of proteoglycans (PG) released into culture media and those present in chondrocyte aggregate (by a specific PG radioimmunoassay) showed a maximum production on Days 3 to 5 of culture, then the production decreased and stabilized (from Day 10 to the end of culture). The observed difference between the amounts of PG in aggregates after 20 d and those after 2 h of culture demonstrated that PG neosynthesis did occur during cultivation. This conclusion was supported by other results obtained by [¹⁴C]glucosamine incorporation in chondrocyte aggregates. Moreover, the aggregate fresh weight related to cell number (appreciated by DNA assay) increased significantly with culture duration. Three-dimensional chondrocyte culture represents an interesting model: chondrocytes were differentiated morphologically as well as biosynthetically and synthesized a new cartilage matrix. This work was suported by grant 3.4529.81 from FRSM, Belgium.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.