STAT1 is Constitutively Activated in the T/C28a2 Immortalized Juvenile Human Chondrocyte Line and Stimulated by IL-6 Plus Soluble IL-6R

Abstract

T/C28a2 immortalized juvenile human chondrocytes were employed to determine the extent to which activation of Signal Transducers and Activators of Transcription-1 (STAT1) occurred in response to recombinant human interleukin-6 (rhIL-6) or rhIL-6 in combination with the soluble IL-6 receptor (sIL-6R). Two forms of STAT1, STAT1A and STAT1B, were identified on SDS-PAGE and western blotting with anti-STAT1 antibody. Western blotting revealed that STAT1 was constitutively phosphorylated (p-STAT1). Although incubation of T/C28a2 chondrocytes with rhIL-6 (50 ng/ml) increased p-STAT1A by Δ=22.3% after 30 min, this percent difference failed to reach significance by Chi-square analysis. Similarly, no effect of rhIL-6 (Δ=+10.7%) on p-STAT1B was seen at 30 min. In contrast, although the combination of rhIL-6 plus sIL-6R had no effect on p-STAT1A, rhIL-6 plus sIL-6R increased p-STAT1B by Δ=73.3% (p<0.0001) after 30 min compared to the control group and by Δ=56.7% (p<0.0001) compared to rhIL-6 alone. Janex-1, a Janus kinase-3-specific inhibitor (100 μM) partially reduced the effect of rhIL-6 on p-STAT1B by Δ=27.7% (p<0.05). The results of this study showed that STAT1A/STAT1B was constitutively activated in T/C28a2 chondrocytes. Although rhIL-6 increased p-STAT1B to a small extent, the combination of rhIL-6 plus sIL-6R was far more effective in stimulating STAT1B phosphorylation compared to controls or rhIL-6 alone. These data support the likelihood that although JAK3-mediated activation of STAT1 in T/C28a2 chondrocytes may involve the IL-6/IL-6R/gp130 pathway, these results indicated that STAT1 activation in response to IL-6 preferentially involved IL-6 trans-signaling via sIL-6R.

Keywords: Chondrocyte; Cytokine; Rheumatoid arthritis.

Figure 1

Activation of JAK/STAT by IL-6. The membrane-bound IL-6R (mIL-6R) pathway (far left), canonical IL-6/IL-6R/gp130 pathway (middle), and “non-canonical” IL-6 trans -signaling pathway (far right) is shown where IL-6 interacts with sIL-6R. The canonical IL-6/IL-6R/gp130 pathway is known to activate JAK/STAT and SAPK/MAPK signaling [3]. The JAK/STAT pathway activated by IL-6/IL-6R/gp130 may also activate the SAPK/MAPK pathway via a “cross-talk” mechanism (→?→) [3]. JAK/STAT could also be activated by IL-6 via mIL-6R in cells lacking the gp130 subunit [53]. Under these conditions activated STAT proteins may also initiate SAPK/MAPK signaling (↓?).

Figure 2

Western blot of U-STAT1. T/C28a2 chondrocytes were incubated for 0 or 30 min with 1) no additions; 2) DMSO; 3) 4) rhIL-6 (50 ng/ml); 5) rhIL-6+Janex-1 (100 μM) or rhIL-6 plus sIL-6R (30 ng/ml). The western blot shows A) U-STAT1A (band C); U-STAT1B (band D); B) β-actin; D) Quantified U-STAT1A and U-STAT1B. Lane 1, No additions, 0 min; Lane 2, DMSO, 0 min; Lane 3, rhIL-6+Janex-1, 0 min; Lane 4, No additions, 30 min; Lane 5, DMSO, 30 min; Lane 6, rhIL-6+Janex-1, 30 min; Lane 7, rhIL-6, 30 min; Lane 8, rhIL-6+sIL-6R, 30 min.

Figure 3

Western blot of p-STAT1. T/C28a2 chondrocytes were incubated for 0 or 30 min with 1) no additions; 2) DMSO; 3) 4) rhIL-6 (50 ng/ml); 5) rhIL-6+Janex-1 (100 μM) or rhIL-6 plus sIL-6R (30 ng/ml). The western blot shows A) p-STAT1A (band C); p-STAT1B (band D); B) β-actin; D) Quantified p-STAT1A and p-STAT1B. Lane1, No additions, 0 min; Lane 2, DMSO, 0 min; Lane 3, rhIL-6+Janex-1, 0 min; Lane 4, No additions, 30 min; Lane 5, DMSO, 30 min; Lane 6, rhIL-6+Janex-1, 30 min; Lane 7, rhIL-6, 30 min; Lane 8, rhIL-6+sIL-6R, 30 min.