Indeed, YKL-40 mRNA expression was robustly activated in tissues surrounding the site of turpentine injection and its induction was accompanied by a very strong induction of both IL-1 and IL-6 mRNAs (Fig. 1A)
RelB mRNA expression was also strongly activated in vivo by turpentine in an IL-1-dependent model of irritant-induced sterile inflammation (Fig. 4E).
Since p65 subunit of NF-κB and STAT3 were previously implicated in cytokine-induced expression of YKL-40 (8, 34), we knocked down their expression in human astrocytes
Surprisingly, knockdown of p65 had no effect on YKL-40 mRNA expression (Fig. 3A), but did drastically diminish the expression of IL-8 mRNA, which is p65-dependent (Fig. 3B).
Knockdown of either RelB or p50 significantly diminished cytokine-induced YKL-40 mRNA expression, whereas knockdown of p65, cRel and p52 had no effect (Fig. 4A). This finding implicates both RelB and p50 in YKL-40 regulation.
Cytokine stimulation facilitated the binding of protein complexes to the proximal NF-κB element in vitro, which super-shifted with anti-p65, anti-RelB and anti-p50 antibodies (Fig. 5D), indicating that the proximal NF-κB site can bind both p65/p50 and RelB/p50 complexes.
These data suggests that RelB regulates expression of YKL-40 in response to proinflammatory cytokines, such as IL-1 and TNF, which induce canonical activation of RelB/p50 complexes.
Cumulatively, these data suggest that in cells expressing relatively low levels of YKL-40, such as astrocytes, cytokine-driven RelB promotes YKL-40 induction in vitro and in vivo.
These experiments were performed in U373 glioma cells, which similarly to human and mouse astrocytes, upregulate expression of both YKL-40 and RelB in response to IL-1 and OSM, and this cytokine-induced expression is diminished by the knockdown of p50 and RelB (Suppl. Fig. 4).
Although RelB and p50 did not bind to the YKL-40 promoter in unstimulated U373 cells, binding of RelB and p50 was apparent in cytokine-treated cells (Fig. 5B), suggesting that RelB/p50 complexes directly regulate YKL-40 expression.
We conclude that RelB/p50 complexes can directly bind to the proximal NF-κB site of the YKL-40 promoter and are essential for the cytokine-induced YKL-40 expression.
These data suggest that in addition to STAT3 activation, OSM enhances YKL-40 expression by promoting formation of the RelB/p50 complexes, which bind to the YKL-40 promoter
In agreement with the mutational analysis that showed the importance of the proximal NF-κB site (Fig. 3D), strong protein binding to the proximal, but not distal, NF-κB site of the YKL-40 promoter was induced by IL-1 alone or together with OSM (Fig. 5C).
NF-κB and STAT3 are the major transcription factors activated by IL-1 and OSM, respectively
OSM and IL-1 efficiently regulated YKL-40 expression via STAT3 (Fig. 3A) and OSM promoted the recruitment of IL-1-induced p50 to the YKL-40 promoter (Fig. 5B).
Downregulation of STAT3 (Fig. 3C) dramatically diminished IL-1/OSM-induced YKL-40 mRNA expression (Fig. 3A).
Similarly, RelB mRNA expression was also up-regulated by IL-1 in mouse astrocytes (Fig. 4D).
Indeed, p50/RelB complexes were formed in response to IL-1
More importantly, OSM significantly enhanced IL-1-induced formation of the p50/RelB complexes
Interestingly, although human astrocytes constitutively express low levels of RelB, IL-1 induced dramatic RelB protein accumulation in these cells (Fig. 4C).
In contrast to astrocytes and U373 cells, we found that basal expression of YKL-40 and RelB mRNA was very high in primary human chondrocytes and not stimulated by IL-1 or OSM (Supplementary Fig 3).
In these cells, basal expression of YKL-40 is RelB-, p65-, and STAT3-independent, implicating other unknown factors.
Immunization of mice with MOG35-50 peptide resulted in the induction of EAE, and modest upregulation of YKL-40 mRNA expression in the spinal cords of animals (Fig. 1B).
In addition, expression of both IL-1 and IL-6 mRNAs was also enhanced
Constitutively active STAT3 enhanced, whereas dominant-negative IκBα diminished cytokine-responsiveness.
Since TNF also efficiently activates NF-κB, we tested whether, similarly to IL-1, TNF regulates YKL-40 expression by a RelB-dependent mechanism.
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.