Nuclear translocation of STAT3 and NF-κB are independent of each other but NF-κB supports expression and activation of STAT3

    Nuclear translocation of STAT3 and NF-κB are independent of each other but NF-κB supports expression and activation of STAT3 Antons Martincuks, Katarzyna Andryka, Andrea K¨uster, Hildegard SchmitzVan de Leur, Michal Komorowski, Gerhard M¨uller-Newen PII: DOI: Reference:

S0898-6568(17)30006-2 doi: 10.1016/j.cellsig.2017.01.006 CLS 8821

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

13 May 2016 31 December 2016 4 January 2017

Please cite this article as: Antons Martincuks, Katarzyna Andryka, Andrea K¨ uster, Hildegard Schmitz-Van de Leur, Michal Komorowski, Gerhard M¨ uller-Newen, Nuclear translocation of STAT3 and NF-κB are independent of each other but NFκB supports expression and activation of STAT3, Cellular Signalling (2017), doi: 10.1016/j.cellsig.2017.01.006

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ACCEPTED MANUSCRIPT Nuclear translocation of STAT3 and NF-B are independent of each other but NF-B supports expression and activation of STAT3

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Antons Martincuks1, Katarzyna Andryka2, Andrea Küster1, Hildegard Schmitz-Van de Leur1, Michal Komorowski2, Gerhard Müller-Newen1*

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Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany 2

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* Corresponding author: [email protected]

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Systems Biology of Biochemical Signalling, Laboratory of Modelling in Biology and Medicine, Institute of Fundamental Technological Research, Polish Academy of Sciences, Pawinskiego 5b, 02-106 Warszawa, Poland

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ACCEPTED MANUSCRIPT Abstract NF-κB and STAT3 are essential transcription factors in immunity and act at the interface of the

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transition from chronic inflammation to cancer. Different functional crosstalks between NF-κB and STAT3 have been recently described arguing for a direct interaction of both proteins. During

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a systematic analysis of NF-κB/STAT3 crosstalk we observed that appearance of the subcellular distribution of NF-κB and STAT3 in immunofluorescence heavily depends on the fixation

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procedure. Therefore, we established an optimized fixation protocol for the reliable simultaneous analysis of the subcellular distributions of both transcription factors. Using this protocol we

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found that cytokine-induced nuclear accumulation of NF-κB or STAT3 did not alter the subcellular distribution of the other transcription factor. Both knockout and overexpression of

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STAT3 does not have any major effect on canonical TNFα-NF-κB signalling in MEF or HeLa cells. Similarly, knockout of p65 did not alter nuclear accumulation of STAT3 in response to IL6. However, p65 expression correlates with elevated total cellular levels of STAT3 and STAT1

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and supports activation of these transcription factors. Our findings in MEF cells argue against a

Keywords

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functional interactions.

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direct physical interaction of free cellular NF-κB and STAT3 but point to more intricate

STAT3, NF-κB, signal transduction, nuclear translocation, crosstalk

Abbreviations CFP, cyan fluorescent protein; IL, interleukin; JAK, Janus kinase; MEF, murine embryonic fibroblasts; NF, nuclear factor; NLS, nuclear localization signal; PBS, phosphate-buffered saline; R, receptor; s, soluble; STAT, signal transducer and activator of transcription; TNF, tumour necrosis factor; WT, wild-type; YFP, yellow fluorescent protein

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ACCEPTED MANUSCRIPT 1 Introduction The signal transducer and activator of transcription 3 (STAT3) belongs to a family of

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transcription factors consisting of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and

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STAT6 which are involved in the regulation of numerous cellular processes [1]. STAT3 is

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ubiquitously expressed and has the most pleiotropic functions among all STATs [2]. The family of IL-6-type cytokines signals through the cytokine receptor gp130 and these cytokines are

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among the most potent physiological activators of STAT3 [3]. Like other members of the STAT

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family, STAT3 signalling involves tyrosine phosphorylation by Janus kinases (JAKs) or other tyrosine kinases, dimerization and nuclear translocation. In the nucleus, STAT3 binds to DNA

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and regulates target gene expression. Upon dephosphorylation [4], STAT3 dissociates from DNA

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and exits the nucleus via CRM1-mediated export into the cytoplasm, where it becomes available for reactivation [5-7]. Aberrant STAT3 signalling has been shown to be a key factor in

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oncogenesis, chronic inflammation and autoimmune conditions, defining STAT3 as a promising therapeutic target [8, 9].

Nuclear factor kappa B (NF-κB) is a collective name for several dimeric transcription factors composed of members of the Rel family of DNA-binding proteins comprising p65 (RelA), RelB, c-Rel, NF-κB1 (p50 and its precursor p105) NF-κB2 (p52 and its precursor p100). A heterodimer of p50 and p65 subunits is the most abundant form and often simply referred to as NF-κB. In this form, NF-κB is a crucial regulator of many physiological and pathophysiological processes including control of innate and adaptive immune responses, inflammation and cancer progression [10]. Similar to STAT3, NF-κB relays signals from activated receptors in the plasma membrane

to the nucleus, where it regulates gene expression. However, in resting cells NF-κB is maintained in an inactive form by sequestration in the cytoplasm through interaction with inhibitors of kappa

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ACCEPTED MANUSCRIPT B (IκB). Canonical NF-κB activation by TNFα involves phosphorylation and proteasomal elimination of IκBα uncovering a nuclear localization signal (NLS) of NF-κB. [11].

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Consequently, NF-κB accumulates in the nucleus and regulates the expression of target genes

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[12]. NF-κB-dependent re-synthesis of IκB proteins as part of a negative feedback loop and

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additional mechanisms then lead to NF-κB dimer dissociation and CRM1-dependent nuclear export [13, 14].

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In recent years, a functional crosstalk between STAT3- and NF-κB-mediated signal transduction pathways has been described as having a central role in cancer progression [15, 16] and acute

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phase response in the liver [17]. Numerous types of interactions between NF-κB and STAT3

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have been observed, including an overlapping set of target genes, physical association,

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competition or cooperation at gene promoters and others [15, 18]. However, a coherent picture of how both factors can directly influence each other’s signalling has not emerged. On the one

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hand, STAT3 was required for the RhoA-induced NF-κB gene transcription, while nuclear accumulation of p65 subunit was reported to be impaired in STAT3 deficient cells upon both Rho and TNFα stimulation [19, 20]. In contrast, an augmented nuclear p65 presence and increased expression of typical NF-κB target genes upon STAT3 depletion was observed in A549 lung cancer cells [21], glioblastoma cells [22] and primary mouse pneumocytes [23]. Little is known about the effect of p65 deletion or overexpression on STAT3 activity. To resolve these issues, we have investigated the effects of conditional knockouts or overexpression of STAT3 and NF-κB transcription factors on each other. For this purpose, we have chosen cultured mouse embryonic fibroblasts (MEF) as a well-established system for studying the impact of gene knockouts on basic cellular processes. Although total knockout of STAT3 led to early embryonic lethality in mice [24], conditional gene targeting has been

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ACCEPTED MANUSCRIPT successfully used to study STAT3 functions in specific tissues and cells [25, 26] and STAT3 knockout MEF have been established [27]. Furthermore, p65 deficient MEF have also been

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reported to proliferate and thrive in culture [28]. In this report, we have focused on the

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involvement of NF-κB p65 in IL-6-induced STAT3 activation, nuclear accumulation and gene

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expression, as well as on the reciprocal role of STAT3 in canonical TNFα-induced NF-κB signalling using p65-deficient and STAT3-deficient MEF along with inducible expression of the

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transcription factors in HeLa cells.

2 Materials and Methods

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2.1 Cytokines, cytokine receptors, antibodies and chemicals.

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Recombinant human IL-6 and soluble IL-6 receptor alpha (sIL-6Rα) were prepared as described

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previously [29, 30]. Recombinant human TNFα was obtained from Peprotech (#300-01A, USA). Anti-pY701-STAT1 (#7649), anti-pY705-STAT3 (#9131), anti-pS536-p65 (#3033), anti-pS32IκBα (#2859), anti-STAT3 (#9139), anti-p65 (#4764), anti-IκBα (#4814 Cell Signaling, USA), anti-lamin A/C (sc-6215), anti-STAT1 (sc-346) anti-GAPDH (sc-32233, Santa Cruz Biotechnology, USA) and anti-dsRed (#632496, Clontech Laboratories, Mountain View, USA) antibodies were used for immunodetection. Anti-rabbit, anti-goat and anti-mouse horseradish peroxidase conjugated antibodies (DAKO, Germany) were used as secondary antibodies. 2.2 Plasmid constructs The pMOWS expression vectors were kindly provided by Prof. Jürgen Scheller, Heinrich-Heine University, Germany, as described in [31]. The expression vector containing human p65 was kindly provided by Dr. Marcus W. Covert [32] and was used for cloning of pMOWS-p65–YFP

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ACCEPTED MANUSCRIPT by PCR. S276A point mutant of p65 was generated with PCR based QuikChange® XL SiteDirected Mutagenesis Kit according to manufacturer's instructions (Agilent Technologies, USA)

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antisense 5'- GCTCCCGGTCGGCAGGCCGCCGCAGC-3'.

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using the following primers: sense 5'-GCTGCGGCGGCCTGCCGACCGGGAGC-3' and

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2.3 Cell culture and transfection

HeLa, wild-type murine embryonic fibroblasts (MEF), STAT3-deficient (STAT3-/-) (generous

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gift from Prof. Valeria Poli, University of Turin, Italy, described in [27]) and p65-deficient (p65-

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/-) (generous gift from Prof. Lienhard Schmitz, Justus-Liebig-University, Germany, described in [28]) MEF were cultivated in Dulbecco’s modified Eagle’s medium (DMEM)/GlutaMAX

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(Gibco, Paisley, UK) supplemented with 10% FCS (Life Technologies GmbH, Germany) and

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1% penicillin/streptomycin (Sigma-Aldrich, USA). STAT3-deficient MEF stably transfected with (WT)STAT3-CFP-YFP have been described earlier [7] and were additionally supplemented

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with 300 μg/ml hygromycin B (Invivogen, France). MEF p65 -/- cells were retrovirally transduced with p65-YFP or p65(S276A)-YFP fusion proteins using the pMOWS system and supplemented with 1 μg/ml puromycin (Invivogen, France). Inducible HeLa Flp-In T-REx cells were stably transfected for the inducible expression of p65, dsRed (monomer), p65-dsRed (monomer) and STAT3-eGFP proteins using the Flp-In system (Invitrogen, UK) and supplemented with 250 μg/ml hygromycin B and 15 μg/ml blasticidin (Invivogen, France) for selection and 100 μg/ml hygromycin B and 10 μg/ml blasticidin for cultivation. The expression of the encoded protein was induced with 10 ng/ml doxycycline (Sigma-Aldrich, USA) for 24 h. 2.4 Preparation of cell lysates, SDS-PAGE and immunoblotting MEF were cultured on six-well plates and stimulated as described in the figure legends. For total cell lysates cells were lysed on ice for 30 minutes in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 6

ACCEPTED MANUSCRIPT 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM NaF, 15% glycerol). For subcellular fractionation, cells were resuspended in hypotonic lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM

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NaCl, 3 mM MgCl2) and centrifuged at 2,000 RPM for 10 min to collect the supernatant

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(cytosolic fraction). The resulting pellet was washed three times in nuclear isolation buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40) to remove cytoplasmic

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waste, resuspended in nuclear lysis buffer (20 mM Tris-HCl pH 7.5, 280 mM NaCl, 10 mM NaF,

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2% Triton X-100) and centrifuged again at 13,000 RPM for 10 min to collect the supernatant representing the nuclear fraction. All lysis buffers were supplemented with phosphatase/protease

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inhibitors (1 mM sodium orthovanadate, 0.5 mM EDTA, 0.25 mM phenylmethylsulfonylfluoride (PMSF), 5 μg/ml aprotinin, 2.5 μg/ml leupeptin). Protein concentrations of the lysates were

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determined by the Bio-Rad protein assay (Bio-Rad, Germany). Protein lysates were separated by 10% SDS-PAGE and transferred to a PVDF membrane by semi-dry blotting with subsequent

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immunodetection using the indicated antibodies. Bound secondary antibodies were detected by pCA-ECL chemiluminescence and signals analysed with Fujifilm MultiGauge v3.2 software. 2.5 Immunofluorescence

Cells were grown on 18 mm glass coverslips and stimulated as indicated. After washing with PBS++ (2.7 mM KCl pH 7.5, 137 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM MgCl2, 0.1 mM CaCl2) and fixed either with 3.7% PFA or ice-cold 100% methanol at room temperature for 20 minutes for one-step fixation, or with 3.7% PFA at room temperature and subsequent icecold 100% methanol at –20°C for 10 minutes. Afterwards, the cells were permeabilized with PBS++ containing 0.1% Triton X-100 (PBST++) for 5 minutes, quenched with 50 mM NH4Cl in PBST++ and blocked with PBST++ containing 5% BSA (Serva, Germany). Cells were labeled with rabbit p65 antibody and mouse STAT3 antibody. The manufacturer Cell Signaling

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ACCEPTED MANUSCRIPT recommends paraformaldehyde fixation for p65 antibody (#4764) and methanol fixation for STAT3 antibody (#9131) during sample preparation. Alexa Fluor® 555 anti-rabbit and Alexa

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Fluor® 488 anti-mouse antibodies (A-31572 and A-21202; Thermo Fischer Scientific, USA)

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were used as secondary antibodies. Nuclei were stained with DRAQ5™ (Biostatus, UK).

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2.6 Confocal microscopy

Confocal imaging was performed with a Zeiss LSM 710 confocal microscope (Zeiss, Germany)

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using a NA 40x/1.1 water immersion objective. Alexa Fluor® 555 fluorescence was excited

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using the 561 nm laser, emission was detected in the range of 562-650 nm. Alexa Fluor® 488 signal was excited using the argon 488 nm laser, emission was detected in the range of 493-550

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nm. DRAQ5™ nuclear staining was detected using the 633 nm line of the Red Helium-Neon

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laser and a 661-759 nm detection range. Mean nuclear fluorescence of A555-stained p65 and

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A488-stained STAT3 was calculated using Mathworks MATLAB®. 2.7 RNA preparation and quantitative RT-PCR MEF were stimulated as indicated and total RNA was extracted according to the manufacturer's instructions using RNeasy Mini Kit (Qiagen, Netherlands). Afterwards, 1 μg of total RNA was reverse transcribed using random hexamers (Sigma-Aldrich, USA) and Omniscript Kit (Qiagen, Netherlands). qPCR was performed with Sybr-Green reaction mix (#QT650-02; Bioline, USA) using Rotorgene (Qiagen, Netherlands). Expression was normalized to the housekeeper gene mGUSB (#QT00176715, Qiagen, Germany). Following primer sequences were used for amplification of cDNAs: 5`- CCG CGG GCA CCT TTC-3` forward and 5`- TTG ACG CTC AAC GTG AAG AAGT - 3` reverse for socs3, 5`- CTC CCC CTA CCA GCT TAC CT -3` forward and 5`- TAG GGC AGC TCA TCC TCT GT - 3` reverse for ikba.

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ACCEPTED MANUSCRIPT 2.8 Statistical analysis The relative cDNA expression ratio was calculated by the ∆∆Ct method [33]. Quantification of

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immunofluorescence, imunoblotting and RT-PCR data was plotted using GraphPad Prism 6

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software and statistically evaluated by Student’s t-test. A P value of less than 0.05 was

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considered as significant.

3 Results

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3.1 Canonical nuclear translocation of STAT3 and NF-κB are independent of each other. We previously showed IL-6-induced nuclear accumulation of endogenous STAT3 using

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methanol fixation and immunofluorescence staining of MEF [34]. However, paraformaldehyde fixation has been reported to be better suited for visualization of NF-κB p65 localization by

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immunofluorescence [35]. The choice of the processing method is most important for appearance of the immunostaining and the interpretation of the results may vary depending on the protocol used [36]. In order to find optimal conditions to analyse both p65 and STAT3 subcellular localization in parallel, the two conditions of fixation were compared along with combined paraformaldehyde and methanol treatment (Fig. 1A). MEF were treated for 30 min with TNF or IL-6 to induce nuclear translocation of endogenous p65 or STAT3, respectively, or left untreated. TNFα-induced nuclear accumulation of p65 was clearly visible in paraformaldehyde fixed cells but hardly detectable upon methanol fixation (Suppl. Fig. 1A,B). However, paraformaldehyde fixation is not suited for the analysis of cytokine-induced nuclear accumulation of STAT3 because already in unstimulated cells the bulk of STAT3 appeared nuclear (Suppl. Fig. 1A). In order to resolve these problems, we applied a combined protocol of paraformaldehyde fixation

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ACCEPTED MANUSCRIPT followed by methanol treatment and after immunofluorescence staining assessed the subcellular localization of both factors by confocal microscopy (Suppl. Fig. 1C). Under these conditions,

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STAT3 shows uniform distribution in resting state and nuclear accumulation upon IL-6, but not TNFα treatment, as expected. In turn, p65 is cytoplasmic in unstimulated cells and enters the

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nucleus after TNFα but not after IL-6 stimulation. These results correlate with subcellular

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fractionation analysis (Suppl. Fig. 1D) and demonstrate that a joint paraformaldehyde and

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methanol fixation protocol is useful for antibodies with different fixation preferences for indirect

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immunofluorescence.

Next, we determined whether cytokine-induced nuclear translocation of one factor affects subcellular distribution of the other one. Physical and functional interaction between p65 and

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STAT3 have been reported in cells after prolonged IL-6 or TNFα treatment [37]. For this reason, we stimulated MEF with TNFα and IL-6 for up to 8 hours and analysed activation and nuclear

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presence of both factors by subcellular fractionation (Fig. 1B). Immunoblotting of cytoplasmic and nuclear extracts revealed that TNFα treatment resulted in IκBα phosphorylation and degradation as expected as well as p65 nuclear accumulation after 30 minutes with less pronounced nuclear presence after 4 and 8 hour stimulation time. However, STAT3 activation and subcellular localization was not affected. Similarly, IL-6 induced STAT3 phosphorylation and nuclear translocation without having any significant effect on p65 activation and nuclear presence. Furthermore, these observations were confirmed by simultaneous visualization and quantification of endogenous p65 and STAT3 subcellular distribution under the same conditions via indirect immunofluorescence (Fig. 1C). Therefore, it appears evident that nuclear accumulation of STAT3 or NF-κB does not affect the subcellular distribution of the other transcription factor in wild-type MEF, arguing against a physical association of the two proteins.

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ACCEPTED MANUSCRIPT 3.2 Absence of STAT3 has no significant effect on canonical TNFα-induced NF-κB activation, nuclear translocation and target gene expression.

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In wild-type MEF, upon TNFα treatment serine 536 phosphorylation of p65 peaked after 5

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minutes of stimulation, while IκBα was degraded after 15 minutes and returned to normal levels

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at 60 minutes time point (Fig. 2A), representing canonical NF-κB signalling [38]. It has been reported that STAT3 ablation or blockade affects NF-κB signalling [21-23]. Using STAT3

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knockout MEF, we analysed the impact of STAT3 deletion on canonical TNFα-induced NF-κB

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activation. STAT3-/- cells were stimulated with TNFα for indicated time points and compared with similarly treated WT MEF. As seen in Fig. 2A, STAT3-deficient cells showed essentially the same pattern of p65 Ser-536 phosphorylation kinetics, IκBα Ser-32 phosphorylation and

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degradation in response to TNFα stimulation. Cells deficient in endogenous p65 served as a negative control and showed lower total IκBα and, interestingly, also lower STAT3 levels.

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Analysis of GAPDH levels demonstrated equal loading of all lanes. Next, we assessed canonical NF-κB target gene ikba expression changes upon TNFα treatment. As shown by quantitative real time PCR, wild-type and STAT3 deficient fibroblasts have comparable TNFα-induced ikba mRNA fold change levels (Fig. 2B). Moreover, in p65 deficient MEF ikba mRNA upregulation was impaired and stable transfection of p65-YFP fusion protein in these cells reversed the effect, confirming p65 involvement in ikba expression. Finally, we compared p65 nuclear translocation in WT and STAT3-/- MEF using confocal microscopy and subcellular fractionation approaches. In addition to activation, p65 nuclear entry was unaltered after 30 minutes of TNFα stimulation in STAT3-deficient cells compared with WT MEF, as seen by immunofluorescence (Fig. 2C) and immunoblotting of cytoplasmic and nuclear

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ACCEPTED MANUSCRIPT fractions (Fig. 2D). Taken together, our data show that STAT3 knockout has no major effect on canonical TNFα-stimulated NF-κB signalling in MEF.

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3.3 Absence of NF-κB p65 subunit has no influence on IL-6-induced STAT3 activation, but

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negatively affects total STAT3 levels.

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To analyse the influence of p65 on canonical STAT3 signalling, we stimulated wild-type and p65 deficient cells with IL-6 for 30 minutes and assessed STAT3 tyrosine 705 phosphorylation

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via immunoblotting. Both phosphorylation and total levels of endogenous STAT3 were

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decreased in p65 knockout cells compared to wild-type (Fig. 3A). Detection of GAPDH verified equal protein loading in both blots. Subsequently, we analysed expression of the STAT3 target

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gene socs3 upon p65 deletion. As expected, IL-6 treatment led to approximately 10-fold increase

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in socs3 mRNA in wild-type MEF and this effect was absent in STAT3-/- cells. In turn, p65

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knockout led to an almost 2-fold decrease in socs3 upregulation probably due to lower total STAT3 levels. Stable expression of STAT3-CFP-YFP in STAT3-/- cells complemented the lack of endogenous STAT3 as expected. However, in p65-/- MEF reconstituted with p65-YFP socs3 mRNA upregulation in response to IL-6 was significantly increased compared to wild-type cells (Fig. 3B). Lastly, to assess the influence of p65 knockout on STAT3 nuclear import, we stimulated cells with IL-6 for 30 minutes and detected STAT3 nuclear accumulation by confocal microscopy and immunoblotting of cytoplasmic and nuclear lysates. Despite lower total STAT3 fluorescence signal, p65-/- MEF showed prominent nuclear accumulation of endogenous STAT3 similar to wild-type cells (Fig. 3C). The same pattern was observed in nuclear extracts. (Fig. 3D). These findings show that upon p65 knockout IL-6-induced STAT3 activation is unaffected but total STAT3 levels are decreased resulting in diminished expression of STAT3 target genes upon IL-6 stimulation.

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ACCEPTED MANUSCRIPT 3.4 NF-B stimulates expression and activation of STAT1 and STAT3, but not STAT5 To further explore the observation of lower total STAT3 levels in p65-/- MEF (Fig. 2A, 3A), we

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analysed total protein levels of STAT3, as well as STAT1 and STAT5. In p65-deficient cells

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total expression of STAT1 and STAT3, but not STAT5, was downregulated (Fig. 4A). To

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examine whether lower levels of STAT3 and STAT1 were not an off-target effect of genetic manipulations in p65-/- cells, we stably transfected these cells with constructs expressing p65-

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YFP and the transcriptionally defective p65(S276A)-YFP mutant [39]. Upon expression of p65-

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YFP total amounts of STAT1 and STAT3 recovered (Fig. 4A and B) and were comparable to expression levels in wild-type MEF, confirming the involvement of p65 in the regulation of

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STAT1 and STAT3 expression (Fig. 3B). Moreover, expression of p65(S276A)-YFP did not

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restore STAT1 and STAT3 levels, suggesting the requirement of p65 transcriptional activity in regulation of STAT1/3 expression. Interestingly, total STAT5 levels were not affected by these

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manipulations (Fig. 4A and B).

Since we could rescue total STAT3 and STAT1 levels upon p65-YFP reconstitution in p65-/cells, which also resulted in an increase in socs3 mRNA upon IL-6 treatment compared to wildtype MEF (Fig. 3B), we studied the effect of p65 overexpression on expression and activation of endogenous STAT3. For this purpose a HeLa Flp-In T-REx system was utilized, which allows for conditional expression of the gene of interest upon doxycycline induction. The cells were stably transfected with a plasmid encoding p65-dsRed. Doxycycline induced and non-induced cells were either stimulated with IL-6 or left unstimulated and cytosolic and nuclear fractions were prepared. Immunoblotting revealed a considerable increase in tyrosine phosphorylation and nuclear accumulation of STAT3 in cells overexpressing p65-dsRed upon IL-6 stimulation (Fig. 4C). Moreover, endogenous STAT1 shows an atypical activation and nuclear presence upon IL-6

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ACCEPTED MANUSCRIPT treatment when p65 is overexpressed, confirming the influence of p65 levels on both STAT3 and STAT1 signaling. However, in contrast to MEF, p65-dsRed overexpression does not

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substantially alter total STAT3 and STAT1 protein amounts in HeLa cells, as judged by

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immunoblotting of cytoplasmic fractions (Fig. 4C). In order to prove, that the p65-dsRed overexpression effect on STAT3 overactivation is p65 dependent, we generated two additional

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inducible HeLa Flp-In T-REx cell lines stably transfected with p65 or dsRed. These cells were

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stimulated with IL-6 or left unstimulated with or without doxycycline induction and total cell lysates were analysed by immunoblotting. Only cells expressing p65 STAT3 showed increased

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IL-6-induced phosphorylation upon doxycycline induction (Suppl. Fig. 2A), confirming that the effect observed in Fig. 4B is not due to dsRed induction or doxycycline treatment itself. Taken

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together, inducible p65 overexpression in HeLa cells leads to an increased STAT3 activation and uncharacteristic STAT1 nuclear presence upon IL-6 stimulation. Of note, upon STAT3

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overexpression in HeLa cells IκBα phosphorylation and degradation, as well as p65 nuclear translocation were not affected (Suppl. Fig. 2B). 4 Discussion

STAT3 and NF-κB are pleiotropic transcription factors that are involved in various physiological processes and both are constitutively activated in numerous cancers most often due to activation of upstream signalling pathways in response to autocrine and paracrine signals present within the tumour microenvironment [16]. In recent years, several forms of crosstalk between NF-κB and STAT3 pathways have been described [15, 18]. Here we show that STAT3 has no direct influence on canonical TNFα-induced NF-κB signalling, while NF-κB p65 subunit positively affects STAT3 and STAT1 signalling.

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ACCEPTED MANUSCRIPT We first defined optimal sample preparation conditions for simultaneous visualization of both p65

and

STAT3

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cultured

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immunofluorescence

using

combined

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paraformaldehyde/methanol treatment. The choice of the processing method is most important

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for appearance of the immunostaining and the interpretation of the results may vary depending on the protocol used [36]. In our hands, NF-κB p65 subunit nuclear accumulation upon TNFα

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treatment is clearly detectable after paraformaldehyde fixation but hardly visible after methanol

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fixation (Fig. 1A, Suppl. Fig.1A and B). Our findings are in agreement with previously published data that methanol treatment results in a perinuclear localization of p65 in human dendritic cells

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upon stimulation [35]. Paraformaldehyde preparation also preserved the intracellular and

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FasL proteins in liver cells [40].

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membrane structures better than methanol and resulted in a more accurate localization of Fas and

In turn, several reports describe the visualization of STAT3 nuclear translocation by microscopy

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have using different methods of fixation [34, 41, 42]. Our data demonstrates that paraformaldehyde fixation leads to a predominant nuclear localization of endogenous STAT3 both in resting and IL-6-stimulated cells, while methanol fixation and combined protocols lead to a more uniform distribution of STAT3 in unstimulated cells and a significant increase in nuclear STAT3 after cytokine treatment (Fig. 1A, Suppl. Fig.1A). These observations could explain previously reported prominent nuclear localization of endogenous STAT3 in both resting and activated states in different cell lines [42], which is contradictory to live-cell imaging of ectopically expressed STAT3 fluorescent fusion proteins showing predominant cytoplasmic localization of STAT3 in resting cells and rapid nuclear accumulation upon stimulation [34, 43]. Paraformaldehyde fixation has been shown previously to cause misinterpretation of the subcellular localization of plant proteins causing dissociation of these proteins from metaphase

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ACCEPTED MANUSCRIPT chromosomes [44]. While paraformaldehyde preserves structural elements at the expense of reduced antigen accessibility, fixation with methanol can disturb structural integrity of the cells

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[45]. We found a combined paraformaldehyde/methanol fixation protocol to be optimal for

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parallel p65 and STAT3 visualization using antibodies with different fixation preferences. In line with our observations, combined treatment has been reported previously to be more useful for

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proper visualization of transmembrane and soluble cytoplasmic proteins than paraformaldehyde

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or methanol fixation alone [46, 47]. Using combined fixation and subcellular fractionation approaches, we demonstrate that canonical activation and nuclear entry of STAT3 and NF-κB

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are independent of each other (Fig. 1B and D). Our observations from MEF do not reproduce previously observed interactions between the two factors [37], indicating that previously

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observed different crosstalk forms of STAT3 and NF-κB p65 subunit upon TNFα or IL-6

dependent.

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stimulation are not universally applicable, but rather cell-type and extracellular environment

To assess the potential role of STAT3 in canonical NF-κB signalling, we compared TNFαinduced pathway activation in wild-type and STAT3-/- cells. Our results demonstrate that the absence of STAT3 has no significant effect on total p65 levels, p65 phosphorylation on serine 536, IκBα phosphorylation and degradation, as well as subsequent p65 nuclear translocation and ikba gene upregulation (Fig. 2A-D). At least five kinases converge on p65 phosphorylation at serine 536 [48], which is one of the most extensively studied p65 posttranslational modifications [49], suggesting that all molecular steps upstream of p65 phosphorylation remained unaffected

upon STAT3 deletion. In contrast to previously published data [20], the absence of STAT3 had no detrimental effects on TNFα-induced nuclear translocation of NF-κB p65 and target gene expression, as demonstrated by optimized immunostaining protocol and subcellular fractionation

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ACCEPTED MANUSCRIPT experiments (Fig. 2C-D). IκBα is a classical NF-κB target gene, which has is linked with p65 in a regulatory feedback loop [50]. Deletion of p65, but not STAT3, had detrimental effects on ikba

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expression and this effect can be rescued by p65 reconstitution, confirming the dependence of

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IκBα expression on NF-κB p65 subunit (Fig. 2B). Since the regulation of several other target genes has been reported to require both p65 and STAT3 DNA association [51-53], genes other

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than ikba may have been influenced by STAT3 deletion. However, proper ikba upregulation

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indicates that canonical NF-κB signalling is intact in STAT3-/- cells. Furthermore, overexpression of STAT3 also showed no effect on p65 activation and nuclear translocation in

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HeLa cells (Suppl. Fig. 2B). These data indicate that STAT3 has no direct influence on canonical NF-κB signalling upon TNFα stimulation, confirming further that previously published

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observations of STAT3 silencing being beneficial for NF-κB activation in cancer or primary cells [21-23] are not universal, but rather cell-type or stimulus specific, along with the impact of

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oncogenesis or the extracellular microenvironment. Moreover, separate or combined activation of p65 and STAT3 in MEF did not result in any detectable physical association of the two factors in coimmunoprecipitation experiments (data not shown), in contrast to previously published observations in coronary artery smooth muscle cells [54]. Knockout of endogenous p65 in MEF led to downregulation of total STAT3 and STAT1 levels, but not STAT5, and this effect was rescued in p65-YFP-complemented p65-/- cells (Fig. 4A and B) confirming the involvement of p65 in controlling total STAT3 levels [55, 56]. Serine phosphorylation of the p65 subunit affects the activity of NF-κB and its interaction with coregulators [57]. Phosphorylation of p65 at serine 276 regulates a subset of NF-κB target genes with specific cis-acting elements in their promoters, such as those encoding IL-6, IL-8, Gro-β, and ICAM-1 [57], as well as allowing for binding of CREB-binding protein (CBP)/p300 [58].

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ACCEPTED MANUSCRIPT Our data revealed, that serine 276 substitution to alanine in p65 led to a similar decrease in STAT1 and partially STAT3 amounts as observed in p65-deficient cells, indicating that

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transcriptional activity of p65 is involved in regulation of STAT1 and STAT3 expression.

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Despite lower total amounts of protein, cytokine-induced nuclear import of STAT3 remained

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intact in p65-/- cells (Fig. 3B,C) suggesting that IL-6 induced signalling itself was less affected. Interestingly, reconstitution of p65-/- cells with p65-YFP led to an increased socs3 upregulation

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in response to IL-6 (Fig. 3D). Several reports suggest a positive association between NF-κB transcriptional activity and STAT3 activation. In the absence of tumour suppressor p53, both

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NF-κB and STAT3 were constitutively activated and depleting the expression of p65 led to

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suppression of STAT3 constitutive activation [55]. Similarly, suppression of NF-κB activity by

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p65R siRNA in Sirt1-null MEF reduced mitochondrial function and the expression of STAT3 [56]. In turn, in human hypopharyngeal epithelium cells acidic-bile salts induced substantial

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increase in total p65 amounts, which was subsequently correlated with STAT3 upregulation [59]. Moreover, in our hands inducible p65-dsRed overexpression in HeLa cells led to an increased STAT3 activation and nuclear accumulation along with abnormal STAT1 phosphorylation and nuclear presence (Fig. 4C). Under normal conditions in HeLa, as well as other cancer-derived cell lines, IL-6-type cytokines do not induce a significant nuclear accumulation of STAT1 [60]. Yet, in our hands inducible increase of total p65 amount led to a substantial nuclear presence of both activated STAT3 and STAT1 upon IL-6 treatment. Therefore we conclude that p65 supports expression and activation of STAT3 and STAT1, which requires further analysis. STAT3 and STAT1 have been demonstrated to separately associate with NF-κB in the cytoplasm with different effects [37, 61] and recent report demonstrated that higher expression of NFκB in rheumatoid arthritis patients significantly correlated with increased STAT1 and STAT3 amounts

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ACCEPTED MANUSCRIPT in peripheral leukocytes [62]. While both NF-κB and STAT3 signalling pathways, but not MAPKs, were dysregulated by interferons in STAT1-null macrophages, overactivation of

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STAT3 was the key player in downregulation of NF-κB signalling [63]. Additionally, deficiency

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of one of the major NF-κB activating receptors TLR4 led to enhanced STAT1-driven Th1 differentiation and suppressed STAT3-driven Th17 expansion [64], whereas IL-27, a crucial

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cytokine for bridging innate and adaptive immunity, has been shown to rely on all three

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transcription factors for its proper signalling [65]. Taking all this into account, a triple collaboration of STAT1, STAT3 and NF-κB in immunological and pathological processes is

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worth exploring further. Taken together, our results suggest that TNFα-induced canonical NF-κB signalling does not rely on STAT3, while expression of the NF-κB p65 subunit positively

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Conclusions

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correlate with total STAT3 and STAT1 levels and augments STAT3/STAT1 activation.

Taken together, our results suggest that STAT3 does not interfere directly with canonical TNFαinduced NF-κB signalling. However, expression of NF-κB positively correlates with total STAT3 and STAT1, but not STAT5, levels and supports STAT3 and STAT1 activation. Furthermore, our study demonstrates that the fixation procedure has a strong influence on the appearance of the subcellular distribution of STAT3 and NF-κB having important implications for the interpretation of published data. An improved immunostaining protocol for reliable staining of both NF-κB and STAT3 with antibodies preferring different fixation protocols and visualization by fluorescence microscopy is provided.

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ACCEPTED MANUSCRIPT Conflict of interest

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The authors disclose no potential conflicts of interest. All authors approved the final article. Acknowledgments

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This work was supported by the Studienstiftung des deutschen Volkes (to AM) and the

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‘Immunohistochemistry and Confocal Microscopy Facility’, a core facility of the

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Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University. MK and KA were funded by the Polish Ministry of Science and

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Higher Education under IUVENTUS PLUS grant IP2012016572. KA was supported by the Erasmus+ programme. The authors would like to thank Natalie Rinis for generation of the p65-

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dsRed HeLa Flp-In T-REx stable cell line, Thomas Wilhelm for help with qRT-PCR and Oxana

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Khamidova for help with immunofluorescence image quantification.

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ACCEPTED MANUSCRIPT Figure legends Figure 1: Analysis of STAT3 and NF-κB canonical activation. (A) Quantification of nuclear

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p65 and STAT3 via indirect immunofluorescence following 3.7% paraformaldehyde (PFA), methanol (MetOH) or combined (PFA+MetOH) fixation (means ± SD of app. n=100 cells per

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sample). MEF were stimulated with 10 ng/ml TNFα or 20 ng/ml IL-6 with 500 ng/ml sIL-6Rα for 30 minutes, fixed either with 3.7% PFA, methanol or combined 3.7% PFA and methanol

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treatment, permeabilized and stained using specific p65 and STAT3 antibodies, followed by incubation with secondary Alexa Fluor-555 and Alexa Fluor-488 conjugated antibodies

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respectively. ***p < 0.0005. *p < 0.05. n.s. = not significant. (B) Analysis of cytokine-induced STAT3 and p65 nuclear accumulation by cell fractionation. Wild-type MEF were stimulated

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with 20 ng/ml TNFα or 10 ng/ml IL-6 with 500 ng/ml sIL-6Rα for 30, 240 and 480 minutes or left untreated. Afterwards, cytoplasmic and nuclear extracts were prepared and immunoblotted with indicated antibodies. GAPDH served as cytoplasmic marker, lamin A/C served as nuclear

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fraction loading control. Upper band represents lamin A, lower band represents lamin C. (C)

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Simultaneous visualization of p65 and STAT3 subcellular localization following TNFα treatment. Left: Cells were stimulated with 20 ng/ml TNFα for indicated time points or left

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untreated, incubated with primary p65 and STAT3 antibodies, followed by immunostaining with Alexa Fluor-555 and Alexa Fluor-488 antibodies, respectively, as well as DRAQ5 nuclear marker. Right: Nuclear presence of p65 and STAT3 was quantified using MATLAB software (means ± SD of app. n=100 cells per sample). ***p < 0.0005. **p < 0.005. *p < 0.05. (D) Simultaneous visualization of p65 and STAT3 subcellular localization following IL-6 treatment. Cells were stimulated with 10 ng/ml IL-6 with 500 ng/ml sIL-6Rα for indicated time points and samples were prepared, visualized (left) and quantified (right) similarly to Fig.1C. Scale bars represent 10 μm. Figure 2: NF-κB activation, target gene expression and nuclear translocation in wild-type, STAT3-/- and p65-/- MEF upon TNFα stimulation. (A) Analysis of p65 serine 536 phosphorylation and IκBα phosphorylation and degradation. Top: cell lines were stimulated with 20 ng/ml TNFα for 5, 15 and 60 minutes or left untreated. Total cell lysates were prepared and immunoblotted with the indicated antibodies. GAPDH immunodetection served as a loading control. Bottom: Phospho(S536)-p65 and total IκBα signals from three independent

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ACCEPTED MANUSCRIPT immunoblotting experiments were quantified using Fujifilm MultiGauge v3.2 software. ***p < 0.0005. **p < 0.005. *p < 0.05. n.s. = not significant. (B) IκBα mRNA expression upon TNFα treatment. Wild-type MEF, STAT3-/-, p65-/- cells, as well as p65-/- cells stably transfected with

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a fluorescent p65-YFP construct were treated with 10 ng/ml TNFα for 45 minutes. Whole cellular mRNA was isolated, and the expression of ikba was measured by RT-qPCR. Expression

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was normalized to the housekeeper protein mGUSB. Data are representative of three

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independent experiments. ***p < 0.0005. n.s. = not significant. (C) Subcellular localization of p65 and STAT3 following 30 minutes of 20 ng/ml TNFα stimulation. Left: Indicated cell lines

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were fixed with combined PFA/methanol treatment, incubated with primary p65 and STAT3 antibodies, followed by immunostaining with Alexa Fluor-555 and Alexa Fluor-488 antibodies,

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respectively, as well as DRAQ5 nuclear marker. Scale bars represent 10 μm. Right: nuclear presence of p65 was quantified using MATLAB software (means ± SD of app. n=100 cells per sample). ***p < 0.0005. n.s. = not significant. (D) Analysis of p65 nuclear accumulation by cell

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fractionation. Wild-type and STAT3-/- MEF were stimulated with 20 ng/ml TNFα for 30

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Figure 3: STAT3 activation, nuclear translocation and target gene expression in wild-type, p65-/- and STAT3-/- cells upon IL-6 stimulation. (A) Analysis of STAT3 tyrosine 705 phosphorylation. Cell lines were stimulated with 10 ng/ml IL-6 and 500 ng/ml soluble IL-6R (sIL-6Rα) for 30 minutes or left untreated. Total cell lysates were prepared and immunoblotted with the indicated antibodies. GAPDH immunodetection served as a loading control. (B) Increase in SOCS3 mRNA expression upon IL-6 treatment. Wild-type MEF, STAT3-/-, p65-/cells, as well as STAT3-/- MEF stably transfected with STAT3-CFP-YFP and p65-/- cells stably transfected with fluorescent p65-YFP construct were treated with 20 ng/ml IL-6 and 500 ng/ml soluble IL-6R (sIL-6Rα) for 30 minutes. Whole cellular mRNA was isolated, and the expression of socs3 was measured by RT-qPCR. Expression was normalized to the housekeeper protein mGUSB. Data are representative of three independent experiments. *p < 0.0005. ***p < 0.0005. (C) Subcellular localization of p65 and STAT3 following 30 minutes of 10 ng/ml IL-6 and 500 ng/ml soluble IL-6R (sIL-6Rα) stimulation. Left: indicated cell lines were fixed with combined PFA/methanol treatment, incubated with primary p65 and STAT3 antibodies, followed by

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ACCEPTED MANUSCRIPT immunostaining with Alexa Fluor-555 and Alexa Fluor-488 antibodies, respectively, as well as DRAQ5 nuclear marker. Scale bars represent 10 μm. Right: nuclear presence of STAT3 was quantified using MATLAB software (means ± SD of app. n=100 cells per sample). ***p <

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0.0005. n.s. = not significant. (D) Analysis of STAT3 nuclear accumulation by cell fractionation. Wild-type and p65-/- MEF were stimulated with 10 ng/ml IL-6 and 500 ng/ml soluble IL-6R

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(sIL-6Rα) for 30 minutes, followed by cytoplasmic and nuclear extract preparation and

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immunoblotting with STAT3 antibodies. Lamin A/C served as nuclear fraction loading control, GAPDH served as cytoplasmic marker.

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Figure 4: Effect of p65 presence on STAT1, STAT3 and STAT5 expression. (A) Analysis of total STAT1, STAT3 and STAT5 levels upon p65-YFP reconstitution. Total cell lysates of WT,

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p65 -/- and p65 -/- MEF stably transfected with p65-YFP or p65(S276A)-YFP were prepared and immunoblotted with the indicated antibodies. (B) Quantification of total STAT1, STAT3 and STAT5 levels from cell lines analyzed in Fig.4A. Immunoblots from three independent

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experiments were quantified using Fujifilm MultiGauge v3.2 software. *p < 0.05. **p < 0.005.

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***p < 0.0005. n.s. = not significant. (C) STAT3 activation and nuclear translocation of STAT1 and STAT3 after p65-dsRed overexpression. HeLa Flp-In T-REx cells stably transfected with

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p65-dsRed fluorescent fusion protein were left untreated or induced with 10 ng/ml doxycycline for 24 h to induce the expression of the transgene. Subsequently, cells were stimulated with 20 ng/ml IL-6 and 500 ng/ml soluble IL-6R (sIL-6Rα) for 30 minutes and cytosolic and nuclear extracts were prepared and analysed by immunoblotting with the indicated antibodies. Antibodies against GAPDH and lamin A/C were used as cytoplasmic and nuclear loading controls respectively.

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ACCEPTED MANUSCRIPT Highlights Reliable parallel detection of p65 and STAT3 subcellular distribution

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Subcellular distribution of NF-κB and STAT3 is independent of each other

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STAT3 deletion or overexpression does not affect canonical TNFα-NF-κB signalling



Deletion of p65 reduces expression of STAT3 and STAT1



Overexpression of p65 increases STAT3 and STAT1 activation in response to IL-6

AC CE P

TE

D

MA

NU

SC

RI

PT



40