Synergistic effect of IL10+CpG stimulation on proliferation is Myc independent 60

et al., 2000; Hermeking et al., 2000). Therefore, the involvement of endogenous Myc induc-tion in IL10+CpG stimulainduc-tion mediated proliferainduc-tion was accessed. Immunoblotting 2 h after IL10+CpG stimulation revealed a more than 2-fold increase of Myc protein levels compared to unstimulated Myc^lowcells (Figure 20A). To estimate the effect of this Myc increase on pro-liferation and CDK4 expression, a transient knockdown of MYC by siRNA was performed

Figure 20: Endogenous Myc induction is dispensable for IL10+CpG stimulation induced pro-liferation.

(A) Immunoblot of Myc^lowand Myc^highcells after MYC siRNA transfection. 24 h after transfection cells were stimulated with IL10+CpG for 2 h. Knockdown efficiency (Myc/tubulin) relative to non-target (scrb) control cells was calculated using Image J. (B) Relative cell numbers of IL10, CpG and IL10+CpG stim-ulated Myc^lowand untreated Myc^highcells 72 h after MYC knockdown and 48 h after stimulation. Stimuli were added every 24 h. Mean±SD of three independent experiments is shown. (C+D) Relative cell numbers 48 h after Myc inhibition by 60µM 10058-F4 in stimulated Myc^lowcells (C) and unstimulated Myc^highcells (D). Mean±SD of three experiments is shown. (E) qRT analysis of relativeCDK4 expression 24 h after 10058-F4 treatement of simultanous stimulated Myc^lowand unstimulated Myc^highcells. Gene expression was normalized ondrosophila ACT42Aand DMSO treated unstimulated Myc^lowcells. One representative out of three experiments is shown.(^?p < 0.05,^??p < 0.01,^???p < 0.001).

in Myc^lowand Myc^highcells. This knockdown reduced Myc levels of IL10+CpG stimulated cells back to the bases of unstimulated Myc^lowcells (Figure 20A). The reducedMYC expres-sion only affected IL10+CpG stimulation induced cell proliferation about 10 to 20 % after 48 h and more importantly did not block the synergistic effect of IL10+CpG stimulation (Figure 20B). However, a reduction of cell proliferation was observed in Myc^highcells at a knockdown about 40 %. These results were confirmed by inhibition of Myc-Max dimerization by the small molecule inhibitor 10058-F4 (Follis et al., 2008). Incubation with 10058-F4 of Myc^lowcells 3 h prior to stimulation, reduced IL10+CpG stimulation mediated cell doubling, but was not able to block the additional effect of IL10+CpG stimulation on cell doubling com-pared to CpG stimulation alone (Figure 20C). In contrast, a complete block of Myc induced proliferation was observed after 10058-F4 treatment (Figure 20D). This proliferation block was accompanied by a decreased CDK4 expression in Myc^highcells, while the IL10+CpG stimulation mediated CDK4 expression increase was largely unaffected (Figure 20E).

Considering both, Myc knockdown and inhibition experiments, it can be concluded that Myc is only partially involved in IL10+CpG stimulation induced proliferation. Therefore it is pro-posed that the synergistic effect of IL10 and CpG on CDK4 expression and proliferation is mainly driven by other factors than Myc.

3.5.5 IL10+CpG stimulation induced proliferation is NF-κB and STAT3 dependent IL10 and CpG have been shown to activate STAT3 and NF-κB in P493-6 Myc^lowcells, respectively (Figure 6). Combined stimulation with both factors activated the same pathways in Myc^lowcells (Figure 21). Within one hour CpG and IL10+CpG stimulation induced

Figure 21: IL10+CpG stimulation synchronously activates STAT3 and NF-κB signaling in

p65 phosphorylation accompanied by a decrease in IκBα, while both, IL10 and IL10+CpG stimulation, induce STAT3 phosphorylation. In contrast none of these pathways was active in Myc^highcells. Since NF-κB and STAT3 are important mediators of normal and transformed B cell proliferation (Dinget al., 2007; McCarthyet al., 2012) their involvement in IL10+CpG stimulation mediated proliferation was further analyzed.

First, cell doubling after NF-κB or STAT3 pathway inhibition was accessed (Figure 22A).

To inhibit NF-κB signaling two inhibitors against IKK, called ACHP and MLN120B, were used. Incubation with ACHP completely blocked cell proliferation, while the synergistic effect of IL10+CpG stimulation vanished after MLN120B treatment. Same reduction of cell doubling of IL10+CpG to only CpG stimulated levels could be observed after JAK inhibition by using the small molecule inhibitors Ruxolitinib and Pyridone 6. Importantly, Myc^highcell proliferation was only slightly effected by ACHP and Pyridone 6 treatment and not by MLN120B or Ruxolitinib. As an increase of CDK4 expression was thought to be involved in IL10+CpG stimulation induced proliferation, its expression was analyzed after ACHP and Ruxolitinib treatment (Figure 22B). Like observed for cell doubling experiments, ACHP treatment completely blockedCDK4 expression in stimulated Myc^lowcells, but not in Myc^highcells, while Ruxolitinib reduced IL10+CpG stimulation induced expression to CpG stimulation alone.

Figure 22: IL10+CpG stimulation mediated proliferation is NF-κB and JAK/STAT depen-dent.

(A) Relative cell numbers of stimulated Myc^lowand unstimulated Myc^highcells after IKK or JAK inhibi-tion. Cells were preincubated for 3 h with IKK inhibitors (7µM ACHP, 5µM MLN120b) or JAK inhibitors (1µM Ruxolitinib, 1µM Pyridone 6), stimulated twice with IL10 and/or CpG as described before and total cell number was counted after 48 h. Data represent mean±SD of three independent experiments.

Two-way ANOVA (Bonferroni’s posttest) results are shown. (B) Relative CDK4 expression of stimu-lated Myc^lowand unstimulated Myc^highcells measured by qRT PCR 24 h after IKK and JAK inhibitor treatment. Expression was normalized ondrosophila ACT42Aexpression and unstimulated DMSO treated Myc^lowcells. Mean expression values and SD of three independent experiments and One-way ANOVA results are shown.(^?p < 0.05,^??p < 0.01,^???p < 0.001).

Figure 23: CDK4 expression is STAT3 and p65/RELA dependent.

(A) Immunoblot analysis of STAT3 and p65 afterSTAT3,RELAorSTAT3 andRELAknockdown. GAPDH staining indicates equal protein loading. Figure is taken from MD thesis of Judith Kemper (Kemper, unpub-lished). (B) qRT analysis ofCDK4 mRNA expression of stimulated Myc^lowcells after knockdowns shown in A. Analysis was performed 48 h after knockdown and 24 h after stimulation with IL10 and/or CpG. Results are shown relative to drosophila ACT42A and scrb transfected unstimulated Myc^lowcells. Data represent mean±SD of three independent experiments. Two-way ANOVA (Bonferroni’s posttest) results are shown.

To further support these observations, the involvement of NF-κB/p65 and STAT3 in IL10+

CpG stimulation induced proliferation was analyzed with support from Judith Kemper (Kemper, unpublished). She performed siRNA mediated knockdown of STAT3 and p65 (Figure 23A) and observed significantly reduced cell doubling in IL10+ CpG stimulated cells. To confirm the STAT3 and NF-κB dependent regulation ofCDK4, its gene expression was analyzed after STAT3 and/or p65 knockdown by qRT PCR (Figure 23B).

In line with the above described inhibitor experiments, CDK4 mRNA expression was sig-nificantly reduced after STAT3 or NF-κB knockdown, but double knockdown of both tran-scription factors showed strongest reduction of CDK4 expression in IL10+CpG stimulated cells. In conclusion, synchronous STAT3 and p65 activation synergistically increases the expression of cell cycle regulators, for exampleCDK4, and thereby facilitates cell cycle entry and cell doubling in IL10+CpG stimulated Myc^lowcells.

3.5.6 IL10 and CpG do not influence each other’s STAT3 and p65 activation

While an important role of STAT3 and NF-κB in IL10+CpG stimulated cells was confirmed, the mechanism underlying the synergistic effect of both pathways was still unknown. As described in the introduction, STAT3 and NF-κB signaling can interact by modifying each other’s signaling activity or duration (Liu et al., 2013; Lee et al., 2009). Therefore, pathway activation of NF-κB and STAT3 signaling was monitored over time by immunoblotting and a

Figure 24: Comparable time course of STAT3 and NF-κB activation after single and combined stimulation in Myc^lowcells.

(A-C) Comparison of IκBαdegradation and STAT3 activation after single IL10 and CpG and double stim-ulation of Myc^lowcells. Cells were stimulated with either IL10, CpG or both for indicated time points, followed by immunoblot analysis (A). Equal loading is shown by tubulin and ponceau staining. One rep-resentative blot out of three is shown. Activation of pSTAT3 (Tyr705, B) and degradation of IκBα (C) of immunoblot shown in A were quantified using Image J. (D+E) Nuclear translocation time of p65 (D) and pSTAT3 (Tyr705, E) after IL10, CpG and IL10+CpG stimulation analysed by flow cytometry. Mean±SD of three independent experiments are shown.

followed by slow decrease of the signal. In contrast, IκBαwas degraded after 30 min and 1 h but again increased after 2 h in single as well as IL10+CpG stimulated cells. This time course was confirmed by nuclear staining of p65 and STAT3 by flow cytometry. No difference of p65 (Figure 24D) or STAT3 (Figure 24E) nuclear translocation was observed between 15 min and 8 h.

Therefore, differences in signaling strength or duration as a main reason for the synergistic effect of STAT3 and p65 on CDK4 gene expression are excluded. Instead, is suggested that a combined binding of STAT3 and NF-κB to the CDK4 promoter or a second unknown transcription factor regulated by both pathways is underlying the synergistic effect on gene expression.