IGFBP-6/sonic hedgehog/TLR4 signalling axis drives bone marrow fibrotic transformation in primary myelofibrosis

Abstract

Primary myelofibrosis is a Ph-negative chronic myeloproliferative neoplasm characterized by bone marrow fibrosis and associated with the involvement of several pathways, in addition to bone marrow microenvironment alterations, mostly driven by the activation of the cytokine receptor/JAK2 pathway. Identification of driver mutations has led to the development of targeted therapy for myelofibrosis, contributing to reducing inflammation, although this currently does not translate into bone marrow fibrosis remission. Therefore, understanding the clear molecular cut underlying this pathology is now necessary to improve the clinical outcome of patients. The present study aims to investigate the involvement of IGFBP-6/sonic hedgehog /Toll-like receptor 4 axis in the microenvironment alterations of primary myelofibrosis. We observed a significant increase in IGFBP-6 expression levels in primary myelofibrosis patients, coupled with a reduction to near-normal levels in primary myelofibrosis patients with JAK2V617F mutation. We also found that both IGFBP-6 and purmorphamine, a SHH activator, were able to induce mesenchymal stromal cells differentiation with an up-regulation of cancer-associated fibroblasts markers. Furthermore, TLR4 signaling was also activated after IGFBP-6 and purmorphamine exposure and reverted by cyclopamine exposure, an inhibitor of the SHH pathway, confirming that SHH is involved in TLR4 activation and microenvironment alterations. In conclusion, our results suggest that the IGFBP-6/SHH/TLR4 axis is implicated in alterations of the primary myelofibrosis microenvironment and that IGFBP-6 may play a central role in activating SHH pathway during the fibrotic process.

Keywords: IGFBP-6; MPNs; TLR4; mesenchymal stem cells; myelofibrosis.

Figure 1

IGFBP-6 was modulated in PMF patients. (A) z-score expression levels of IGFBP-6 in healthy, JAK2 wild type, and JAK2V617F mutant PMF patients. (B) Correlation analysis between IGFBP-6 and JAK2 expression levels in the PMF patients. (C) mRNA expression of IGFBP-6 in neutrophils of PMF patients. * p<0.05.

Figure 2

IGFBP-6 induces CAF differentiation in mesenchymal stem cells. (A) HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h were lysed and subjected to immunoblotting using specific antibodies against α-SMA, FAP1, and TGF-β. Protein content was normalized to the housekeeping protein β-actin. The entire assay was made in triplicate, a representative one is shown. Signals from immunodetected bands were semi-quantified by densitometry. (B–D) Statistical analysis of data revealed that α-SMA expression was significantly increased in the HS5 cells IGFBP-6- induced for 24h and 48h (B), FAP1 expression was significantly increased in the HS5 cells IGFBP-6- induced for 24h (C) TGF-β expression was significantly increased in the HS5 cells IGFBP-6- induced for 24h and 48h (D). Data are presented as means ± sem. **p < 0.01 and ***p < 0.001 vs. untreated. (E) qPCR results were obtained for TGF-β in HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h. Relative mRNA expression level normalized with β-actin by using a comparative 2-ΔΔCt method. **p < 0.01.

Figure 3

IGFBP-6 induces the expression of mediators involved in proliferation and migration in HS5 cells. (A–G) Multiplex immunobead assay technology on HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h was performed on a culture medium to determine concentrations of indicated cytokines. Culture medium from untreated cells and treated cells were evaluated. (*P < 0.05) (H) qPCR results obtained for BMP2 in HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h. Relative mRNA expression level normalized with β-actin by using a comparative 2-ΔΔCt method. *** P < 0.001.

Figure 4

Purmorphamine-mediated SMO activation recapitulates IGFBP-6 effects on HS5 cells. (A) qPCR results were obtained for SHH in HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h. Relative mRNA expression level normalized with β-actin by using a comparative 2-ΔΔCt method. **p <0.01; ***p < 0.001. (B) Immunofluorescence analysis were performed on HS5 cells treated with IGFBP-6 at the final concentration of 200 ng/mL, followed by fixing and staining with anti-Phalloidin (green) and anti-Gli1 (red). Nuclei were visualized using DAPI. Immunoreactivity was evaluated considering the signal-to-noise ratio of immunofluorescence (scale bar 20 μm). (C) z-score expression levels of Gli1, Gli2, GPATCH1, and SMO in healthy, JAK2 wild type, and JAK2V617F mutant PMF patients. (D–I) Multiplex immunobead assay technology on HS5 cells exposed or not to purmorphamine was performed on culture medium to determine concentrations of indicated cytokines. Histograms showed a significant increase of MMP9 (D), TIMP (E), CHI3L1 (F), BMP2 (G), OPG (H), and sRANKL (I) after purmorphamine stimulation, as compared to control. **p <0.01; ***p < 0.001.

Figure 5

IGFBP-6 induces TLR4 signalling on HS5 cells. (A) HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h were lysed and subjected to immunoblotting using specific antibodies against TLR4 and TLR3. Protein content was normalized to the housekeeping protein β-actin. The entire assays were made in triplicate, a representative one is shown. Signals from immunodetected bands were semi-quantified by densitometry. (B, C) Statistical analysis of data revealed that TLR4 (B) and TLR3 (C) expression were significantly increased in the HS5 cells IGFBP-6- induced for 24h and 48h. Data are presented as means ± sem. **p < 0.01 and ***p < 0.001 vs. untreated. (D) Immunofluorescence analysis were performed on HS5 cells treated with IGFBP-6 at the final concentration of 200 ng/mL, followed by fixing and staining with anti-pNF-kB (red), anti-IRF3 (red), and anti-YAP1 (green). Nuclei were visualized using DAPI. Immunoreactivity was evaluated considering the signal-to-noise ratio of immunofluorescence (scale bar 20 μm).

Figure 6

IGFBP-6-induced TLR4 signalling is controlled by SHH signalling through SMO. (A) qPCR results obtained for IGFBP-6 in HS5 cells exposed or not to purmorphamine. Relative mRNA expression level normalized with β-actin by using a comparative 2-ΔΔCt method. **P < 0.01 and ***P < 0.001. (B) Immunofluorescence analysis were performed on HS5 cells exposed or not to purmorphamine, followed by fixing and staining with anti-pNF-kB (red), anti-IRF3 (red), and anti-YAP1 (green). Nuclei were visualized using DAPI. Immunoreactivity was evaluated considering the signal-to-noise ratio of immunofluorescence (scale bar 20 μm). (C) HS5 cells exposed to purmorphamine, cyclopamine, or both were lysed and subjected to immunoblotting using a specific antibody against IGFBP-6. Protein content was normalized to the housekeeping protein β-actin. The entire assay was made in triplicate, a representative one is shown. Signals from immunodetected bands were semi-quantified by densitometry. (D) Statistical analysis of data revealed that IGFBP-6 expression was significantly increased after exposure to purmorphamine. Data are presented as means ± sem. **p < 0.01 and ***p < 0.001 vs. untreated. (E) HS5 cells exposed to purmorphamine, cyclopamine, or both were lysed and subjected to immunoblotting using specific antibodies against TLR4 and TLR3. Protein content was normalized to the housekeeping protein β-actin. The entire assay was made in triplicate, a representative one is shown. Signals from immunodetected bands were semi-quantified by densitometry. (F, G) Statistical analysis of data revealed that purmorphamine was able to increase while cyclopamine was able to suppress both TLR4 (F) and TLR3 (G) protein expression levels. Co-treatment with both SMO agonist and antagonist did not affect TLR4 and TLR3 expression levels, as compared to control cell cultures. Data are presented as means ± sem. **p < 0.01 and ***p < 0.001 vs. untreated. (H) HS5 cells exposed to IGFBP-6, cyclopamine, or both were lysed and subjected to immunoblotting using a specific antibody against TLR4. Protein content was normalized to the housekeeping protein β-actin. The entire assay was made in triplicate, a representative one is shown. Signals from immunodetected bands were semi-quantified by densitometry. (I) Statistical analysis of data revealed that TLR4 expression levels were significantly increased after IGFBP-6 stimulation, while a cotreatment with cyclopamine had a reducing effect on TLR4 expression. Data are presented as means ± sem. **p < 0.01 and ***p < 0.001 vs. untreated.