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STAT5 and IL-6 dysregulation in mastocytosis
expression intrinsically promotes IL-6 production. Thus, we analyzed the production of IL-6 at the protein and message levels in various cell lines expressing or not D816V-KIT. In agreement with our previous observations, the HMC-1.2 mastocytosis mast cell line which harbors KIT with D816V plus another missense variant in the juxta membrane domain of KIT (V560G), showed markedly higher IL-6 mRNA synthesis13 (Figure 2A, left panel) and IL-6 release than HMC-1.1 cells, which express only the monoallelic V560G-KIT variant (Figure 2A, right panel).
The mastocytoma mouse mast cell line P815 carrying the homolog to the D816V-KIT variant (D814Y-KIT), also released significantly more IL-6 than primary mouse
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BMMC (Figure 2B). Furthermore, expression of human D816V-KIT in an immortalized mouse mast cell line that lacks KIT,13,19 unlike cells expressing normal KIT or vector alone, released significant amounts of IL-6 into the media (Figure 2C). In aggregate, the results using the various mast cell lines demonstrate an association between expression of D816V-KIT in mast cells and persistent tran- scription and release of IL-6. This may not be restricted to mast cells, since introduction of D816V-KIT by CRISPR in the colorectal carcinoma cell line HCT116 also promoted IL-6 transcription and release (Figure 2D, left and right panels, respectively) when cells were stimulated with PMA and ionomycin, indicating that in these cells D816V- KIT primes HCT116 cells for more robust IL-6 production.
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Figure 2. Cells with D816V-KIT constitu- tively express and release IL-6. (A) IL-6 mRNA expression (left) and IL-6 released into the media (right) by the mastocytosis cell lines HMC-1.1 (with V560G) and HMC- 1.2 (with V560G and D816V) after 2 hours (h) in serum-free media. (B and C) Comparison of IL-6 released into the media by the mouse P815 mastocytoma mast cell line (with D814Y-KIT) compared to normal murine bone marrow mononuclear cells (BMMC) (B), and by the murine mast cell line MCBS-1 (which lacks c-Kit) transfected with human KIT or D816V-KIT compared to MCBS-1 transfected with vector alone (C). (D) Comparison of IL-6 mRNA expression (left) and IL-6 released into the media (right) by the human colorectal carcinoma cell line HCT116 expressing or not D816V- KIT. HCT116 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin overnight. IL-6 mRNA expression was determined by quantita- tive-real-time polymerase chain reaction (q-RT-PCR) and relative expression was cal- culated in relationship to the expression of GAPDH using the DCt method and expressed as fold change compared to HMC-1.1 (A, left), or the HCT116 parental cell line (D). All data (A-D) are the results of three independent experiments done in triplicates. *P=0.01; **P<0.01; ***P<0.001 and ****P<0.0001.
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