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Biochem J. 2005 Aug 1; 389(Pt 3): 723–729.
Published online 2005 Jul 26. Prepublished online 2005 Apr 1. doi:  10.1042/BJ20041636
PMCID: PMC1180722

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK


The IGF-I (insulin-like growth factor-I) signalling pathway responsible for regulation of proteoglycan synthesis in chondrocytes has not been defined and is the focus of the present study. Chondrocytes isolated from normal human articular cartilage were stimulated with IGF-I in monolayer culture or in suspension in alginate. IGF-I activated members of both the PI3K (phosphoinositide 3-kinase) pathway and the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway. The PI3K inhibitors LY294002 and wortmannin blocked IGF-I-stimulated Akt phosphorylation without blocking ERK phosphorylation and this was associated with complete inhibition of proteoglycan synthesis. A decrease in IGF-I-stimulated proteoglycan synthesis was also observed upon inhibition of mTOR (mammalian target of rapamycin) and p70S6 kinase, both of which are downstream of Akt. The MEK (MAPK/ERK kinase) inhibitors PD98059 and U0126 blocked IGF-I-stimulated ERK phosphorylation but did not block the phosphorylation of Akt and did not decrease proteoglycan synthesis. Instead, in alginate- cultured chondrocytes, the MEK inhibitors increased IGF-I-stimulated proteoglycan synthesis when compared with cells treated with IGF-I alone. This is the first study to demonstrate that IGF-I stimulation of the PI3K signalling pathway is responsible for the ability of IGF-I to increase proteoglycan synthesis. Although IGF-I also activates the ERK/MAPK pathway, ERK activity is not required for proteoglycan synthesis and may serve as a negative regulator.

Keywords: Akt, articular cartilage, chondrocyte, insulin-like growth factor-I (IGF-I), mitogen-activated protein kinase (MAPK), proteoglycan synthesis
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular-signal-regulated kinase; IGF-I, insulin-like growth factor-I; IGF-IR, IGF-I receptor; IL-1β, interleukin 1β; IRS, insulin receptor substrate; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAPK/ERK kinase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; Tos-Phe-CH2Cl, tosylphenylalanylchloromethane


IGF-I (insulin-like growth factor-I) is a protein produced in mammals that is found in multiple organs, including the liver, intestine, cartilage, bone and in circulation [1,2]. IGF-I acts systemically as well as locally at the sites of its production, to exert endocrine, paracrine and autocrine effects. The principal actions of IGF-I concern the control of growth and organ size through mitotic and antiapoptotic effects, as well as stimulation of protein synthesis. Consistent with this function, severe growth deficiency has been noted in transgenic animals lacking IGF-I or the type 1 IGF-IR (IGF-I receptor) [3,4].

Since IGF-I is an important growth factor in a number of tissues, the signalling pathways activated upon IGF-I stimulation of different cell types have been well documented [2,5]. IGF-I action results primarily from the activation of the type 1 IGF-IR. The major substrates recruited to IGF-IR after its activation are Shc and members of the IRS (insulin receptor-substrate) family including IRS-1 and IRS-2. After IRS phosphorylation, downstream signalling pathways are activated including the PI3K (phosphoinositide 3-kinase) cascade and ERK (extracellular-signal-regulated kinase), a member of the MAPK (mitogen-activated protein kinase) cascade. Activation of PI3K leads to activation of Akt and p70S6 kinase, downstream serine-threonine protein kinases involved in cell-survival and protein synthesis pathways respectively. Coupling of Grb2 (growth-factor-receptor-bound protein 2) with IGF-IR occurs through IRS-1 and Shc, and leads to the activation of Ras and the Raf/MEK/ERK/MAPK cascade (where MEK stands for MAPK/ERK kinase).

In articular cartilage, IGF-I increases the synthesis of extracellular matrix proteins, most notably collagen and proteoglycan [68]. IGF-I has been shown to be the major stimulator of proteoglycan synthesis present in serum and synovial fluid [9,10] and was originally called serum sulphation factor due to its ability to stimulate sulphate incorporation (a measure of proteoglycan synthesis) by cartilage [11]. Surprisingly, the cell signalling mechanism by which IGF-I stimulates proteoglycan synthesis has not been reported.

Elucidation of the signalling pathway responsible for IGF-I-mediated proteoglycan synthesis by chondrocytes is necessary to understand better the mechanism of IGF-I resistance that has been noted in aging cartilage and during the development of osteoarthritis [12,13]. The present study was designed to determine the role of the two major IGF-I signalling pathways, the PI3K and the ERK/MAPK pathways, in the regulation of proteoglycan synthesis using adult human articular chondrocytes. Cells grown both in monolayer as well as in suspension in alginate were studied. Chondrocyte culture in alginate has been shown to preserve the normal chondrocyte phenotype [14], which can be lost after prolonged culture in monolayer [15]. The results show that key members of both the PI3K and ERK/MAPK pathways are activated by IGF-I treatment of chondrocytes, but that only activity of the PI3K pathway is required for the stimulation of proteoglycan synthesis.



LY294002, PD98059, Tos-Phe-CH2Cl (tosylphenylalanylchloromethane; ‘TPCK’), rapamycin, L-744,832 and pronase were purchased from Calbiochem (San Diego, CA, U.S.A.), wortmannin was from Sigma (St. Louis, MO, U.S.A.) and U0126 was from Promega (Madison, WI, U.S.A.). DMEM (Dulbecco's modified Eagle's medium), Ham's F-12, PBS, antibiotics and fetal bovine serum were purchased from Gibco BRL (Gaithersburg, MD, U.S.A.). Collagenase-P was purchased from Boehringer Mannheim (Germany), Keltone LVCR sodium alginate from Kelco (Chicago, IL, U.S.A.) and [35S]sulphate from Amersham Biosciences (San Francisco, CA, U.S.A.). Phosphospecific and non-phosphospecific antibodies directed to Akt, ERK, Shc, p70S6 kinase, GSK3β, Bad and forkhead were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). IGF-I was a gift from Chiron (Emeryville, CA, U.S.A.).

Chondrocyte isolation and culture

Human ankle articular cartilage was obtained from tissue donors within 48 h of death through the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL, U.S.A.) in accordance with institutional guidelines and review board approval. Only human donors with no prior history of arthritis were used in this study. Each donor specimen was graded for degenerative changes based on the five-point Collins scale, as modified by Muehleman et al. [16], and only samples of grade 0 or 1 were used. Full-thickness cartilage was removed from all surfaces of both the right and left tali and pooled for chondrocyte isolation. Human cartilage slices were digested in a sequential manner with 0.2% pronase for 1 h and then overnight with 0.025% collagenase as described previously [17]. Monolayer cultures using freshly isolated chondrocytes were plated on 6-well plates at 2×106 cells/ml in DMEM/F-12 medium supplemented with 10% (v/v) fetal bovine serum. Plates were maintained for approx. 7 days with feedings every 2 days until they reached 100% confluency before experimental use. Alternatively, freshly isolated chondrocytes were placed in alginate culture in serum-free media supplemented with mini-ITS as described previously and cultured for 4–7 days before treatment with IGF-I [17].

Chondrocyte stimulation and analysis of signalling proteins

Chondrocytes were first serum-starved in DMEM/Ham's F-12 medium for 24 h. Inhibitors were then preincubated for 30 min to 1 h before the addition of IGF-I or an equal volume of vehicle for controls. See Table 1 for a complete list of the inhibitors used and their IC50 values. After IGF-I stimulation, media were removed and the cells were washed once with ice-cold PBS containing 0.1 mM Na3VO4. Cell lysates were immediately prepared by solubilization in cell lysis buffer containing 20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF and 1 μg/ml each of aprotinin, leupeptin and pepstatin. The cells were then scraped, transferred on to microcentrifuge tubes, agitated end-over-end for 30 min at 4 °C, centrifuged at 14000 g for 10 min and the supernatants were removed and measured for total protein (bicinchoninic acid kit; Pierce, Rockford, IL, U.S.A.). In signal-transduction experiments performed on cells cultured in alginate, the cells were isolated from the further-removed matrix by dissolving the alginate beads at room temperature (22 °C) for 10 min in 0.4 ml of 55 mM sodium citrate solution in 0.15 M NaCl containing 1 mM Na3VO4, followed by centrifugation at 200 g for 10 min at 4 °C. The super-natant was removed and the cell-pellet fraction was then solubilized in cell lysis buffer as above. Samples from either monolayer or alginate culture were equalized for total protein, separated by SDS/PAGE [10% polyacrylamide] and transferred on to nitrocellulose for immunoblot analysis using antibodies directed to the phosphorylated forms of the signalling proteins of interest and control non-phosphospecific antibodies. Immunoreactivity was determined with enhanced chemiluminescence (ECL®; Amersham Biosciences).

Table 1
Inhibitors and IC50 values

[35S]sulphate incorporation

Chondrocytes were cultured as monolayers or in alginate and treated with kinase inhibitors and IGF-I as described above. After an overnight incubation with IGF-I, [35S]sulphate was added to the media for a final 4 h of culture. Sulphate incorporation was measured using the Alcian Blue precipitation method and by DNA analysis using Hoescht dye as described previously [12].

Determination of cell death

Cell death was determined using the LIVE/DEAD cell-survival assay (Molecular Probes, Eugene, OR, U.S.A.) as described previously [18]. The percentage of cells that remained alive after treatment was measured in triplicate with at least 100 cells counted for each data point.

Statistical analysis

Results comparing the means between multiple groups were analysed using a one-way ANOVA with a post hoc Bonferroni correction using the Windows-based StatView software (SAS Institute, Cary, NC, U.S.A.). Samples for sulphate incorporation were performed in triplicate, and P<0.05 was considered to be statistically significant for all statistical analyses.


IGF-I activates the PI3K and ERK/MAPK signalling pathways in human articular chondrocytes

Stimulation of chondrocytes established in a confluent monolayer with IGF-I at concentrations ≥12.5 ng/ml stimulated Akt phosphorylation at Ser473 (Figure 1A). Because maximal Akt phosphorylation was seen with 50 ng/ml IGF-I, this concentration was used for further time-course studies. Akt phosphorylation was seen within 5 min and continued to the final 60 min time point (Figure 1B). IGF-I also stimulated the phosphorylation of ERK1/ERK2, which was more transient than Akt. Levels of IGF-I-stimulated phospho-ERK returned to control levels by 30–60 min. For comparison, IL-1β (interleukin 1β)-stimulated phosphorylation of ERK was greater than IGF-I and was maintained at 30 min. However, IL-1 did not stimulate the phosphorylation of Akt (Figure 1B). IL-1 but not IGF-I stimulated the phosphorylation of p38 and c-Jun N-terminal kinase (results not shown). IGF-I stimulated the phosphorylation of the p66 subunit of Shc and downstream targets of Akt including GSK-3β, the forkhead transcription factor, and IGF-I reduced the phosphorylation of the pro-death protein Bad (Figure 1C). IGF-I increased the phosphorylation of p70S6 kinase at Thr389 and Thr421/Ser424 as well as Akt at Thr308 in a time-dependent manner. These findings demonstrate the ability of exogenous IGF-I to stimulate members of both the ERK/MAPK and PI3K/Akt pathways in cultured articular chondrocytes.

Figure 1
IGF-I stimulation activates PI3K and ERK/MAPK signalling pathways in chondrocytes

Effects of PI3K and MAPK pathway inhibitors on signalling in chondrocytes

Before using signalling protein inhibitors to determine which proteins activated by IGF-I were required for the stimulation of proteoglycan synthesis, the specific inhibitors were tested for their ability to block the phosphorylation of members of the PI3K and MAPK pathways in chondrocytes. In addition to monolayer culture, chondrocytes were also tested in suspension culture using the alginate system. The PI3K inhibitor LY294002 [19] at 25 μM blocked IGF-I stimulation of Akt phosphorylation in chondrocytes cultured in monolayer (Figure 2A) or alginate (Figure 2C) without affecting the ability of IGF-I to stimulate the phosphorylation of ERK (Figures 2B and and2D).2D). Similar results were obtained using wortmannin at 50 nM (results not shown). The MEK inhibitor PD98059 [20] at 25 μM blocked IGF-I-stimulated phosphorylation of ERK (Figures 2B and and2D)2D) without inhibiting the ability of IGF-I to stimulate the phosphorylation of Akt (Figures 2A and and2C).2C). Preincubation with 10 μM U0126 also inhibited IGF-I-stimulated phosphorylation of ERK and not Akt (results not shown). Cell survival in the presence of any of the four inhibitors was not reduced compared with controls (results not shown).

Figure 2
Effect of PI3K and MAPK inhibitors on IGF-I stimulation of Akt and ERK phosphorylation in chondrocytes

Rapamycin has been shown to inhibit the mTOR (mammalian target of rapamycin) [21], which is downstream of Akt and upstream of p70S6 kinase [22]. Consistent with this, rapamycin at 5 nM blocked the phosphorylation of p70S6 kinase (Figure 3A) without affecting the phosphorylation of ERK (Figure 3C). The anti-p70S6 kinase antibodies used in these experiments also recognized the p85S6 kinase, but rapamycin did not affect the intensity of this band until a concentration of 20 nM was reached. Phosphorylation of Akt appeared to be greater in cells stimulated with IGF-I after pretreatment with rapamycin compared with cells treated only with IGF-I (Figure 3B). Two different p70S6 kinase inhibitors were also tested, L-744,832 and Tos-Phe-CH2Cl, and neither inhibitor significantly altered IGF-I-stimulated Akt phosphorylation (results not shown).

Figure 3
Effect of mTOR inhibition on IGF-I stimulation of p70S6 kinase, Akt and ERK phosphorylation

Inhibition of PI3K but not MEK blocks IGF-I-stimulated proteoglycan synthesis by chondrocytes

Chondrocytes in monolayer were preincubated with a 25 μM concentration of either LY294002, to inhibit PI3K and subsequent activation of Akt, or PD98059, to inhibit MEK and subsequent activation of ERK, or both the inhibitors together. The cells were then incubated overnight with 50 ng/ml IGF-I followed by the addition of radiolabelled sulphate for the assay of proteoglycan synthesis. Compared with controls maintained for the same time period in serum-free media, addition of IGF-I increased sulphate incorporation to approx. 150% of control (Figure 4A) without a significant change in DNA content, an indicator of cell number (Figure 4B). Pretreatment with the PI3K inhibitor completely blocked IGF-I stimulation of sulphate incorporation, which was statistically significant (P<0.001) when sulphate incorporation was normalized for cell numbers (Figure 4C). Inhibition of MEK had no effect on IGF-I-stimulated sulphate incorporation, whereas a combination of the two inhibitors gave results which were in between those obtained with each inhibitor alone and which were not statistically significant from control. Similar to the results in monolayer culture, inhibition of PI3K also blocked IGF-I-stimulated sulphate incorporation in cells cultured in alginate (P=0.03). In alginate cultures, inhibition of MEK significantly (P=0.009) increased IGF-I-stimulated sulphate incorporation to 140% of control, which was above that achieved with IGF-I alone (120% of control; Figure 4D).

Figure 4
Inhibition of PI3K but not ERK blocks IGF-I-stimulated proteoglycan synthesis in chondrocytes cultured as monolayers or in alginate

The alginate experiments were repeated with a second set of PI3K and MEK inhibitors using cultures from four additional donors with similar results. The PI3K inhibitor wortmannin inhibited IGF-I stimulation of proteoglycan synthesis, whereas the MEK inhibitor U0126 did not (Figure 4E). There was a similar trend for U0126 to increase IGF-I stimulation of proteoglycan synthesis above that of IGF-I alone which was statistically significant (123% of control for IGF-I versus 168% of control for IGF-I+U0126, P=0.002) for synthesis of proteoglycans measured in the cell-associated matrix (Figure 4F).

Inhibition of mTOR and p70S6 kinase decreases proteoglycan synthesis in IGF-I-treated chondrocytes

Inhibition of mTOR with rapamycin significantly decreased IGF-I-stimulated proteoglycan synthesis to a level below control (Figure 5A). A p70S6 kinase inhibitor, Tos-Phe-CH2Cl, was also tested and found to decrease IGF-I-stimulated proteoglycan synthesis below control values, suggesting that inhibition of p70S6 kinase not only blocked IGF-I stimulation but also basal proteoglycan synthesis (Figure 5B). However, when cell survival was evaluated, it was noted that Tos-Phe-CH2Cl concentrations above that which inhibited IGF-I-mediated proteoglycan synthesis (10 μM) caused significant chondrocyte death, whereas inhibition of mTOR with rapamycin did not (Figure 6).

Figure 5
Inhibition of mTOR or p70S6 kinase decreases proteoglycan synthesis in IGF-I-treated chondrocytes
Figure 6
Inhibition of p70S6 kinase results in chondrocyte death


IGF-I activates members of both the PI3K and ERK/MAPK signalling pathways in a number of cell types [2,5] and as shown here, has a similar activity in adult articular chondrocytes. Although both pathways were activated by IGF-I, activation of PI3K, but not ERK was required for the stimulation of chondrocyte proteoglycan synthesis. Chemical inhibition of PI3K, which blocked IGF-I-stimulated phosphorylation of Akt, resulted in complete inhibition of IGF-I-stimulated proteoglycan synthesis. Inhibition of mTOR and p70S6 kinase, which are downstream of Akt, also significantly inhibited proteoglycan synthesis. Since IGF-I-mediated activation of mTOR and p70S6 kinase through the PI3K/Akt pathway plays a key role in regulating protein translation [2325], the results suggest that IGF-I regulation of proteoglycan synthesis in chondrocytes may occur primarily through up-regulation of translational activity. In further support of a significant effect of IGF-I on protein translation, we have not been able to detect a significant effect of IGF-I on aggrecan, decorin or biglycan core protein RNA levels in adult human chondrocytes and did not see a change in aggrecan RNA levels after treatment with IGF-I in the presence of LY294002 or PD98059 (J. D. Cravero and R. F. Loeser, unpublished work).

IGF-I has recently been shown to stimulate the differentiation of chick limb bud mesenchymal cells into chondrocytes through the PI3K pathway and similar to our results, inhibition with LY294002 blocked the ability of IGF-I to stimulate the accumulation of proteoglycan by these cells [26]. Also, similar to our findings, inhibition of ERK activation with PD98059 did not block IGF-I-stimulated proteoglycan synthesis by mesenchymal cells, but instead promoted it [26]. MEK inhibition was also found to increase the expression of chondrocyte matrix genes by chick limb mesenchyme [27]. In alginate cultures, we noted an increase in proteoglycan synthesis when cells were treated with IGF-I after MEK inhibition with PD98059 or U0126. Similar results were obtained using cartilage explants (J. D. Cravero and R. F. Loeser, unpublished work). These results suggest that the Ras/Raf/MEK/ERK/MAPK pathway may negatively regulate IGF-I-stimulated proteoglycan synthesis in chondrocytes. The finding that IL-1β stimulated the phosphorylation of ERK but not Akt would also be consistent with this, since IL-1 is known to be a potent inhibitor of proteoglycan synthesis [28,29]. The mechanism by which ERK activity might inhibit IGF-I-stimulated proteoglycan synthesis is not clear but could involve a negative feedback loop through ERK-mediated serine phosphorylation of IRS-1, which has been shown to inhibit insulin signalling [30,31]. Further studies will be necessary to determine whether this mechanism contributes to inhibition of IGF-I signalling in chondrocytes.

In addition to its role in stimulating proteoglycan synthesis, IGF-I has been shown to promote chondrocyte survival [32]. In other cell types, IGF-I survival signalling involves the activation of the PI3K/Akt pathway, although additional pathways appear to be capable of promoting survival [33,34]. In chondrocytes plated on to collagen, IGF-I stimulation of the ERK/MAPK signalling pathway was shown to prevent apoptosis [35]. Under the conditions used in the present study, significant cell death was not observed with inhibition of either the PI3K pathway or the ERK/MAPK signalling pathway. It is possible that one pathway could compensate when the other is inhibited, but we did not observe cell death when the PI3K and MEK inhibitors were combined, suggesting that alternative survival pathways may be activated. These pathways may include p70S6 kinase since chondrocyte death was observed at concentrations of Tos-Phe-CH2Cl above 10 μM.

Our results demonstrated that inhibition of mTOR by rapamycin increased IGF-I-stimulated Akt phosphorylation without changing ERK phosphorylation. Since mTOR is located directly downstream of Akt, these results suggest that a regulatory feedback mechanism may be active. Inhibition of mTOR significantly decreased proteoglycan synthesis despite the increase in Akt phosphorylation, showing that PI3K activation of Akt alone is not sufficient for IGF-I stimulation of proteoglycan synthesis but that the downstream activity of mTOR as well as p70S6 kinase is also required.

A limitation to the present study was the reliance on chemical inhibitors to define the signalling pathways necessary for IGF-I stimulation of proteoglycan synthesis. However, we used inhibitors that have been well established in the literature and have been commonly used by other investigators. In addition, we tested two different inhibitors for PI3K and MEK and obtained similar results. To substantiate further their use in our studies, we also tested inhibitor specificity by analysing the effects on phosphorylation of key components of the respective signalling pathways. Although a molecular approach using transfection of dominant-negative kinase constructs might seem appropriate to confirm the chemical inhibition results, there are limitations of this approach in experiments using primary adult human chondrocytes. Adult human chondrocytes are difficult to transfect efficiently, and in previous work we had to use co-transfection of promoter-reporter constructs to measure the effects of dominant-negative kinase constructs [36,37]. Since the subject of interest in the present study was proteoglycan synthesis, it was not feasible to use this approach.

In conclusion, the results demonstrated that IGF-I stimulation of proteoglycan synthesis by human articular chondrocytes required the activation of the PI3K/Akt/mTOR/p70S6 kinase signalling pathway but not the activation of the Ras/Raf/MEK/ERK pathway. This information will be useful for further studies, which are necessary to determine the mechanism by which IGF-I signalling declines with aging and during the development of osteoarthritis [12,13].


We thank Dr A. Margulis for procuring donor tissue, and the Gift of Hope Organ and Tissue Donor Network and the donor families for providing the tissue. We also thank C. Pacione for technical assistance. This study was supported by the National Institutes of Health (grant RO1 AG16697).


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