Transcriptional mechanisms underlying sensitization of peripheral sensory neurons by granulocyte-/granulocyte-macrophage colony stimulating factors

Abstract

Background: Cancer-associated pain is a major cause of poor quality of life in cancer patients and is frequently resistant to conventional therapy. Recent studies indicate that some hematopoietic growth factors, namely granulocyte macrophage colony stimulating factor (GMCSF) and granulocyte colony stimulating factor (GCSF), are abundantly released in the tumor microenvironment and play a key role in regulating tumor-nerve interactions and tumor-associated pain by activating receptors on dorsal root ganglion (DRG) neurons. Moreover, these hematopoietic factors have been highly implicated in postsurgical pain, inflammatory pain and osteoarthritic pain. However, the molecular mechanisms via which G-/GMCSF bring about nociceptive sensitization and elicit pain are not known.

Results: In order to elucidate G-/GMCSF mediated transcriptional changes in the sensory neurons, we performed a comprehensive, genome-wide analysis of changes in the transcriptome of DRG neurons brought about by exposure to GMCSF or GCSF. We present complete information on regulated genes and validated profiling analyses and report novel regulatory networks and interaction maps revealed by detailed bioinformatics analyses. Amongst these, we validate calpain 2, matrix metalloproteinase 9 (MMP9) and a RhoGTPase Rac1 as well as Tumor necrosis factor alpha (TNFα) as transcriptional targets of G-/GMCSF and demonstrate the importance of MMP9 and Rac1 in GMCSF-induced nociceptor sensitization.

Conclusion: With integrative approach of bioinformatics, in vivo pharmacology and behavioral analyses, our results not only indicate that transcriptional control by G-/GMCSF signaling regulates a variety of established pain modulators, but also uncover a large number of novel targets, paving the way for translational analyses in the context of pain disorders.

Figure 1

GMCSF- or GCSF- mediated gene pool in the peripheral sensory neurons. (A) Heat map representation of significantly-regulated transcripts showing more than two-fold change (two-tailed t-test assuming equal variance, P with Benjamini and Hochberg False Discovery Rate correction <0.05) following chronic exposure to GMCSF or GCSF in sensory neurons of the DRG. The color scale represents quantile normalized hybridization intensities for each gene. (B) Venn diagram representing the number of genes commonly or differentially regulated by GMCSF and GCSF exposure in DRG neurons. (C) Selected pain-related genes that were significantly regulated (two-tailed t-test assuming equal variance, P with Benjamini and Hochberg False Discovery Rate correction <0.05). Genes commonly (green text) or differently (red text) regulated by GMCSF and GCSF stimulation in sensory neurons are shown. Gene regulation confirmed by qRT-PCR analysis is highlighted in bold text.

Figure 2

Nanostring-nCounter-based validation of selected genes those are up-regulated by GMCSF (A), down-regulated by GMCSF (B), up-regulated by GCSF (C) and down-regulated by GCSF (D) in the sensory neurons. Fold-change expression in the genes is expressed as arithmetic average over 5 housekeeping genes namely Cltc, Gapdh, Gusb, Hprt and Tubb5 in all panels. * P < 0.05, one-way ANOVA followed by Fisher’s LSD Post-hoc analysis.

Figure 3

Direct-interactions-network analysis on GMCSF-mediated gene pool with fold-change between +4 and −4 as compared to control-treated sensory neurons and P-BH < 0.05 (t-test, P with Benjamini and Hochberg False Discovery Rate < 0.05). Genes upregulated and downregulated in GMCSF-dependent manner are marked with red and blue circles, respectively. Please see Additional file 3: Figure S2 for information on legends.

Figure 4

Direct-interactions-network analysis on GCSF-mediated gene pool with fold change between +4 and −4 and as compared to control-treated sensory neurons and P-BH < 0.05 (t-test, P with Benjamini and Hochberg False Discovery Rate < 0.05). Genes upregulated and downregulated in GCSF-dependent manner are marked with red and blue circles, respectively. Please see Additional file 3: Figure S2 for information on legends.

Figure 5

QRTPCR-based validation of GM-CSF-induced regulation of the expression of Rac1, MMP9, Calpain2 and TNFα in vitro as well as in vivo. Gene regulation at 24 h following the start of GMCSF exposure in cultured sensory neurons is shown in (A) and in DRGs at different time points following intraplantar GMCSF injection is shown in (B). * denotes P ≤ 0.05 as compared to vehicle group, One-Way ANOVA followed by Fisher’s LSD Post-hoc analysis, n = 3 mice per group. (C) Schematic representation of the protocol followed to investigate changes in GMCSF-mediated mechanical and thermal hypersensitivity upon inhibition of Rac1 or MMP9 or Calpain 2 or TNFα in vivo.

Figure 6

In vivo validation of GM-CSF transcriptional targets in DRG neurons in the context of nociceptive sensitization evoked by long-term exposure to GMCSF. Changes in GMCSF-mediated mechanical hypersensitivity upon inhibition of Rac1 (A), MMP9 (C), Calpain-2 (E) or TNFα (G) as compared to corresponding vehicle-treated mice are shown. Response frequency to the von Frey filament at 0.16 g force is represented on the Y-axis. Changes in GM-CSF-mediated thermal hypersensitivity upon inhibition Rac1 (B), MMP9 (D), Calpain-2 (F) or TNFα (H) as compared to corresponding vehicle-treated mice are shown. Withdrawal latency in seconds to calibrated radiant heat is represented on the Y-axis. * denotes P ≤ 0.05 as compared to basal values, ^† denotes P ≤ 0.05 relative to corresponding vehicle treated group, One-Way ANOVA with repeated measures followed Fisher’s LSD Post-hoc analysis , n = 6 mice per group.