Function of the chondrocyte PI-3 kinase-Akt signaling pathway is stimulus dependent

Abstract

Objective: The PI-3 kinase-Akt pathway plays a role in cartilage anabolic as well as catabolic processes in response to activation by insulin-like growth factor-1 (IGF-1) and the pro-inflammatory cytokines interleukin-1β (IL-1β) and oncostatin M (OSM). The goal of this study was to determine how PI-3 kinase-Akt signaling regulates these seemingly opposing functions.

Design: Monolayer cultures of primary human articular chondrocytes were treated with IGF-1, IL-1β, OSM, or the combination of IL-1β and OSM in time course experiments. Activation of signaling proteins and MMP production were measured by immunoblotting. Cells were pre-treated with chemical inhibitors to block mitogen activated protein (MAP) kinases, PI-3 kinase, or JAK/STAT pathway activation. Constitutively active Akt1 and Akt3 were expressed to study stimulus-independent activation of Akt.

Results: IGF-1, OSM, and the combination of IL-1β and OSM but not IL-1β alone, stimulated phosphorylation of Akt which was sustained longer with IGF-1. IL-1β plus OSM, but not IGF-1, increased chondrocyte MMP-13 production which was inhibited with either a general PI-3 kinase inhibitor or specific inhibition of the PI-3 kinase-γ isoform. Akt1 or Akt3 activity alone was not sufficient to increase production of MMP-13. IL-1β/OSM induced MMP-13 production required activation of the MAP kinases, JNK and p38, as well as the JAK-STAT pathway which were activated by IL-1β plus OSM but not by IGF-1.

Conclusions: The chondrocyte integrates signals from the PI-3 kinase-Akt pathway with signals from MAP kinases and the JAK-STAT pathway to allow for a differential response to a pro-anabolic (IGF-1) and a pro-catabolic (IL-1β plus OSM) stimulus.

Keywords: Cell signaling; Chondrocyte; Matrix metalloproteinase-13; PI-3 kinase.

Figure 1. Time course of chondrocyte signaling activated in response to IGF-1, IL-1β, and OSM

Normal human chondrocytes were stimulated with IGF (100ng/ml), IL-1β (10ng/ml), OSM (10ng/ml), or IL-1β and OSM together (10ng/ml each) for increasing time points (5–60min). Total cell lysates were immunoblotted for phosphorylated signaling kinases; total kinase blots are shown as loading controls. Representative blots are shown from three independent experiments performed using cells from three different tissue donors.

Figure 2. Densitometric analysis of time course of chondrocyte signaling activated in response to IGF-1, IL-1β, and OSM

The signal of the phosphorylated kinase was normalized to the total kinase loading control and shown as fold-change from untreated control. Data are shown as the average of three independent experiments performed using cells isolated from three different normal tissue donors.

Figure 3. PI-3K, MAPK, and STAT inhibition block IL-1β/OSM induced MMP-13 production

A. Normal chondrocytes were pre-treated with 5µM LY294002, followed by 30 min stimulation (p-Akt blot) or overnight stimulation (MMP-13 blot). Representative blots are shown (n=3 independent experiments using cells from three different tissue donors). MMP-2 is used as a loading control for MMP-13, and total Akt is shown as a loading control for p-Akt. Strong p-Akt in the IGF-1 stimulated cells reduces the ability of the total antibody to recognize Akt. B. Chondrocytes were pre-treated with inhibitors for 30 min and then stimulated with IL-1β/OSM overnight. Conditioned media was collected and immunoblotted for MMP-13. MMP-2 was used as a loading control. C. Densitometric analysis of immunoblots from independent experiments in B (n=6 independent experiments with cells from six unique tissue donors, controls and MAPK inhibitors; n=3 independent experiments using cells from three unique tissue donors, Ruxolitinib). Data are presented as mean ± SD. Because human donors produce variable levels of MMP-13 in response to IL-1β/OSM, densitometry data are normalized to IL-1β/OSM and shown as percent inhibition from the positive control. Treatment (mean ± SD): Control (13.23±10.96); IL-1β/OSM (100.0±0.0); PD+IL-1β/OSM (43.13±22.99); SP+IL-1β/OSM (41.18±24.71); SB+IL-1β/OSM (20.07±16.66); Rux+IL-1β/OSM (10.67±5.71). *=p<0.0001

Figure 4. Over-expression of Akt1 or Akt3 has no effect on IL-1β/OSM induced MMP-13 production

A. Wild type (WT) and constitutively active (CA)-Akt1 were over-expressed in normal chondrocytes, followed by treatment with IL-1β/OSM. Akt 1 constructs contain a hemagglutinin (HA) expression tag, so the HA blot of total lysates is shown to confirm expression of constructs. Representative blots are shown (n=3 independent experiments using cells from three unique tissue donors). B. WT, CA, and dominant negative (DN)-Akt3 were over-expressed in normal chondrocytes, followed by treatment with IL-1β/OSM. Representative blots are shown (n=3 independent experiments using cells from three unique tissue donors). MMP-2 is shown as loading control for conditioned media.

Figure 5. Inhibition of PI-3K-γ significantly reduces IL-1β/OSM induced MMP-13 production

A. Baseline mRNA expression of PI-3K isoforms in chondrocytes from five normal donors (results are presented as individual data points with the mean indicated by the horizontal line). Expression level (mean±SD): TBP (1.00±0.0); PI-3K-α (3.84±2.56); PI-3K-β (3.97±0.42); PI-3K-δ (1.61±0.34); PI-3K-γ (.06±.04). B. Chondrocytes in monolayer were pre-treated with inhibitor (5uM LY or 10uM A66, TGX, CAL, or AS) for 30 min followed by 30 min stimulation with 100ng/ml IGF-1 or 10ng/ml IL-1β+10ng/ml OSM. Representative blots are shown (n=3 independent experiments using cells from three unique tissue donors). C. Chondrocytes in monolayer were pre-treated with inhibitors for 30 min followed by overnight stimulation with 10ng/ml IL-1β+10ng/ml OSM. Representative blots from MMP blots of conditioned media are shown (n=3 independent experiments using cells from three unique tissue donors). D. MMP-13 in conditioned media was quantified by total MMP-13 ELISA. Data are normalized to amount of MMP-13 stimulated by IL-1β/OSM without inhibitors (set to 100%). Data shown are mean ± SD (n= 3 independent normal tissue donors). Treatment (mean±SD): Control (17.99±13.01); IL-1β/OSM (100.0±0.0); LY+IL-1β/OSM (35.02±8.53); A66+IL-1β/OSM (122.3±23.42); TGX+IL-1β/OSM (73.99±32.16); CAL+IL-1β/OSM (74.00±20.07); AS+IL-1β/OSM (41.38±23.54). Control vs. IL-1β/OSM, p=0.0027; IL-1β/OSM vs. LY+IL-1β/OSM, p=0.176; IL-1β/OSM vs. AS+IL-1β/OSM, p=0.0358.

Figure 6. Integrated signaling network of key pathways activated by IGF-1, IL-1β, OSM, and IL-1β/OSM

The integrated signaling network is color-coded to track pathways activated by IGF-1 (green), IL-1β (red), OSM (blue), and IL-1β/OSM (parallel red and blue). Solid lines with arrows indicate pathway activation and kinase phosphorylation by indicated stimulus. The dashed line is to indicate that IL-1β by itself does not stimulate Akt phosphorylation but does when used in combination with OSM. The weight of the lines indicates the relative level of phosphorylation stimulated (eg, IGF-1 is a strong stimulator of Akt phosphorylation and PG synthesis, so those green arrows are weighted heavily). This signaling network illustrates that while IGF-1 and the combination of IL-1β and OSM share activation of the PI-3K-Akt pathway, they have different downstream effects due to differential activation of the JNK and p38 MAP kinases as well as STAT3.