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[Cancer Research 63, 8271-8277, December 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

HIPK2 Regulates Transforming Growth Factor-ß-Induced c-Jun NH2-Terminal Kinase Activation and Apoptosis in Human Hepatoma Cells

Thomas G. Hofmann1, Nicole Stollberg1, M. Lienhard Schmitz2 and Hans Will1

1 Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Hamburg, Germany, and
2 Departement für Chemie und Biochemie, Universität Bern, Bern, Switzerland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase involved in transcriptional regulation and apoptosis. Here we demonstrate that HIPK2 regulates transforming growth factor (TGF) ß-induced c-Jun NH2-terminal kinase (JNK) activation and apoptosis. HIPK2 colocalizes with Daxx, a protein acting in TGF-ß-induced JNK activation and apoptosis, in promyelocytic leukemia (PML) nuclear bodies, and triggers PML-nuclear body disruption and release of Daxx. HIPK2 interacts in vitro and in vivo via its kinase domain with Daxx, and a fraction of Daxx coprecipitates with HIPK2 under physiological conditions. Moreover, overexpression of HIPK2 leads to Daxx phosphorylation, and ectopic expression of HIPK2 activates the JNK signaling pathway, which is enhanced by coexpression of Daxx. HIPK2 signals to JNK via a pathway using Daxx and the mitogen-activated protein kinase kinases MKK4/SEK1 and MKK7. Ectopic expression of HIPK2 and Daxx potentiates TGF-ß-induced apoptosis in human p53-deficient hepatocellular carcinoma cells. Finally, we demonstrate that knockdown of endogenous HIPK2 using RNA interference inhibits TGF-ß-induced JNK activation and apoptosis. Taken together, our findings indicate that HIPK2 participates in the TGF-ß signaling pathway leading to JNK activation and apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth arrest and apoptosis are crucial mechanisms to counteract cellular transformation and carcinogenesis of mammalian cells. HIPK2 is a serine/threonine kinase participating in transcriptional regulation (1 , 2) , growth control (3) , and apoptosis (4 , 5) . Upon treatment with UV-radiation and the chemotherapeutic drug arsenic trioxide, HIPK23 is recruited to PML-NBs, interacts with p53, phosphorylates p53 at serine 46, and triggers apoptosis (4 , 5) .

PML-NBs are nuclear multiprotein complexes thought to function as protein storage pools and post-translational modification platforms. PML and its associated NBs play a role in transcriptional regulation, antiviral response, cellular senescence, tumor suppression, and apoptosis (6, 7, 8) . In APL, PML is fused to the retinoic acid receptor {alpha}, resulting in expression of an oncogenic PML-retinoic acid receptor {alpha} fusion protein, which leads to the disruption of PML-NBs and the development of APL through a multifunctional mechanism. Treatment of APL patients with arsenic trioxide or retinoic acid triggers disease remission, which is paralleled by reformation of PML-NBs (9) .

Daxx was originally identified as an interactor for the CD95 (Fas/APO-1) death receptor, and demonstrated to mediate activation of the MAPK JNK through activation of ASK1 (10 , 11) . Later, Daxx was found in the cell nucleus where it associates with chromatin, centromeres, and PML-NBs, and functions as a transcriptional regulator (12, 13, 14, 15, 16, 17, 18, 19) . ASK1 recruits Daxx from the nucleus to the cytoplasm where both can elicit a cytoplasmic cell death pathway, which is inhibited by HSP27 (20, 21, 22) . It has been demonstrated recently that Daxx also interacts with the cytoplasmic domain of the type II TGF-ß receptor (TGF-ß RII). In analogy to its function in the CD95 signaling pathway, Daxx transmits TGF-ß-induced JNK activation and apoptosis in hepatocytes and B cells (23) . TGF-ß belongs to a superfamily of conserved pleiotropic cytokines, which play a pivotal role in tissue homeostasis, immune cell function, differentiation, cell growth, tumor suppression, and apoptosis (24, 25, 26, 27) . Interestingly, Daxx knockout mice die early during embryogenesis upon induction of apoptosis, demonstrating that Daxx fulfils also an antiapoptotic function (28) .

Here we present evidence that HIPK2 regulates TGF-ß-induced JNK activation and apoptosis in human hepatoma cells independent of p53. Our results provide an explanation of how HIPK2 can suppress cell growth in the absence of p53, and indicate that HIPK2 plays a critical role in regulating TGF-ß-induced JNK activation and apoptosis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfections.
293, 293T, U2OS, and Hep3B (all from American Type Culture Collection) cells were maintained in DMEM/10% FCS/1% (w/v) penicillin/streptomycin/20 mM HEPES buffer. Transient transfections were done using SuperFect (Qiagen, Inc.), FuGene 6 (Roche Molecular Biochemicals), or by standard calcium phosphate precipitation.

Antibodies.
Anti-Daxx (M-112 and H-7), anti-PML (PG-M3), anti-JNK1 (F-3), and JNK2 (D-2) antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-Flag (M2) was obtained from Sigma, anti-HA (12CA5) from Roche Molecular Biochemicals, and the rabbit polyclonal anti-GFP from Clontech. The Erk1/2, phospho-Erk1/2, and the phospho-p38 antibodies were purchased from New England Biolabs. The rabbit polyclonal anti-HIPK2 antibody was described previously (4) .

Expression Constructs.
Human Daxx and human HIPK2 constructs were generated by standard PCR techniques and inserted in pGEX4T1 (Amersham Pharmacia) or pCMV-Tag 2B (Stratagene). All of the PCR generated constructs were confirmed by DNA sequencing. Detailed cloning descriptions can be obtained from the authors on request. Flag-DaxxC was kindly provided by Xiaolu Yang and David Baltimore (California Institute of Technology, Pasadena, CA). Daxx 501–740 was kindly provided by Thomas Sternsdorf (The Salk Institute, La Jolla, CA) and the 2 x SRE-luciferase reporter was a gift from Andrew D. Sharrocks (University of Manchester, Manchester, United Kingdom).

GST Pulldown Assays.
GST-Daxx fusion proteins were expressed in Escheria coli BL21 pLysS cells (Stratagene), purified, and eluted using standard protocols. In vitro translated 35S-labeled proteins were produced by using the TNT kit (Promega). Six to 8 µl of the 35S-labeled proteins were incubated with 3–4 µg of the indicated GST-fusion proteins at 4°C for 2 h in in vitro interaction buffer [150 mM NaCl, 20 mM HEPES (pH 7.5), 0.05% NP40, 2 mM NaF, and 1 mM sodium vanadate] on a rotating wheel, and glutathione-beads were added followed by an additional 1 h of incubation. The beads were washed three times in 750 µl in vitro interaction buffer. Bound proteins were eluted by denaturing the beads in SDS-sample buffer for 5 min at 95°C, followed by SDS-PAGE, Coomassie Brilliant Blue staining, drying, and autoradiography.

Immunofluorescence Stainings.
Immunofluorescence stainings were performed as described (4) . The following primary antibodies were used: mouse anti-PML (PG-M3; 1:50) and rabbit anti-Daxx (M-112; 1:50). Various secondary antibodies were used: Alexa-488-coupled goat {alpha} mouse and Alexa-594-coupled goat {alpha} rabbit (Molecular Probes). DNA was visualized by DAPI or Draq5 staining. Cells were mounted on glass slides and examined either using an Epifluorescence microscope (Axioplan-2; Zeiss) or a confocal laser scanning microscope (LSM510; Zeiss).

Luciferase Assays.
Cells (293) seeded in six-well plates were transfected with 750 ng 2 x SRE-luc (29) or 3 x nuclear factor {kappa}B luciferase reporter gene plasmids and 3 µg of the indicated expression constructs. Total DNA amounts were kept equal in all of the transfections by adding empty pcDNA3 vector. Subsequently, cells were incubated for 24 h in DMEM/0.5% FCS at 37°C. Luciferase activity was measured as published (4) .

Western Blotting and Coimmunoprecipitations.
Cells were lysed in TOTEX buffer [20 mM HEPES (pH 7.9), 350 mM NaCl, 20% (v/v) glycerol, 1% (v/v) NP40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 10 mM NaF, 0.5 mM sodium vanadate, and protease inhibitors] and fractionated by 8.5% or 10% Mini-SDS-PAGE (Bio-Rad), transferred to Immobilon-P (Millipore), and treated as described (4) . Proteins were detected by enhanced chemiluminescence (Western Blot Femto; Pierce). For coimmunoprecipitation of endogenous HIPK2 and Daxx, 2 x 107 U2OS cells were washed in warm PBS and incubated in freshly prepared PBS containing 0.5 mM of the membrane-permeable cross-linker dimethyl-3–3'-dithiobispropionimidate 2-HCl (DTBP; Pierce) for 30 min at 37°C before lysis in TOTEX/100 mM Tris buffer supplemented with 0.1% SDS. Centrifuged lysates were cleared for 1 h with protein A/G agarose beads (Santa Cruz Biotechnology Inc.) at 4°C on a rotating wheel. Equal amounts of lysates were incubated overnight with either 5 µg control rabbit IgG antibody or 5 µg rabbit polyclonal anti-GFP antibodies together with 30 µl protein A/G agarose beads at 4°C on a rotating wheel. The beads were washed three times in 750 µl TOTEX buffer, sedimented by centrifugation, and denatured for 5 min at 95°C in SDS-sample buffer and analyzed by Western blotting. Mapping of interaction domains was done by transfecting 293T cells with the indicated expression constructs. Twenty-four h later cells were lysed [0.1% (v/v) NP40, 10% (v/v) glycerol, 180 mM NaCl, 25 mM NaF, 1 mM sodium vanadate, and protease inhibitors], cell debris was spun down, and supernatants were cleared for 1 h with protein A/G agarose beads, and subsequently immunoprecipitation with 2 µg anti-Flag antibody or 2 µg anti-HIPK2 antibody was done overnight. Precipitates were washed three times in 750 µl lysis buffer, denatured for 5 min at 95°C in SDS sample buffer, and analyzed by immunoblotting.

In Vitro JNK Kinase Assays and Daxx Phosphorylation.
In vitro JNK kinase assays were done as described previously (30) In brief, cells seeded in 6-cm dishes were transfected with 1 µg HA-JNK1, 1 µg Daxx, and 3 µg of Flag-HIPK2 expression constructs. Cells were lysed in NP40 lysis buffer, and the JNK protein contained in the cell lysate was precipitated by the addition of 1 µg of anti-JNK1 (F-3), anti-JNK2 (D-2; for endogenous JNK activation), or anti-HA (for HA-JNK1) antibodies, and 25 µl of protein A/G plus agarose. The precipitate was washed three times in lysis buffer and two times in kinase buffer [20 mM HEPES/KOH (pH 7.4), 1 mM DTT, 25 mM ß-glycerophosphate, and 20 mM MgCl2]. The kinase assay was performed in a final volume of 20 µl kinase buffer containing 2 µg of bacterially expressed GST-c-Jun (5–89) protein, 5 µCi [{gamma}-32P]ATP, and 20 µM ATP. After incubation for 20 min at 30°C, the reaction was stopped by the addition of 5 x SDS loading buffer, separated by SDS-PAGE, fixed, dried, and analyzed by autoradiography.

For in vivo phosphorylation of Daxx, 10-cm dishes of 293 cells were transfected with 10 µg Flag-HIPK2 constructs and 5 µg pcDNA3-Daxx using calcium phosphate precipitation. After 30 h, cells were washed with PBS (37°C) and incubated for 8 h in phosphate-free DMEM supplemented with 1 mCi 32P-ortho-phosphate (NEN). Cells were washed in PBS and lysed in TOTEX buffer. Aliquots of the lysates were analyzed by Western blotting. Daxx was immunoprecipitated for 2 h at 4°C with 2 µg anti-Daxx antibody (M-112) and protein A/G plus agarose beads. The washed immunoprecipitates were denatured in SDS-sample buffer and separated by 8.5% SDS-PAGE. The gels were fixed, dried, and analyzed by autoradiography.

Apoptosis Measurement and TGF-ß Treatment.
Apoptosis was determined by trypan blue exclusion as described previously (4) or by staining Hep3B cells in vivo for 30 min with DAPI (1 µg/ml) containing medium and analyzing 150 cells for nuclear chromatin condensation. Only cells that were round and showed condensed nuclei were considered apoptotic. For TGF-ß treatment, cells were incubated in DMEM/0.1% FCS and subsequently stimulated with 2–4 ng/ml TGF-ß1 (Sigma) for the indicated periods.

Small Interfering RNA (siRNA) Treatment.
Human HIPK2 siRNAs targeting nucleotides 570–589 relative to the first nucleotide in the start codon (sense 5'-CAC CTA CGA GGT CTT AGA G-3'; antisense: 5'-CTC TAA GAC CTC GTA GGT G-3') and 846–865 (sense: 5'-GCA AAA CAA GTT TAG CCC C-3'; antisense: 5'-GGG GCT AAA CTT GTT TTG C-3') were synthesized by Dharmacon. The control luciferase siRNA was kindly provided by T. Heise (Hamburg, Germany). Hep3B cells were seeded in 24-well plates or 6-cm dishes and transfected with the indicated siRNAs using OligoFectamine (Invitrogen) as described (31) . Transfected cells were incubated in DMEM/0.1% FCS overnight and subsequently treated with TGF-ß as indicated.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIPK2 Colocalizes with Daxx in PML-NBs and Releases Daxx from PML-NBs.
We and others have demonstrated recently that HIPK2 is recruited to PML-NBs by PML isoform IV, and that a fraction of endogenous PML-NBs colocalize with endogenous HIPK2-NBs in U2OS cells (4 , 5) . Given the importance of the two PML-NB proteins HIPK2 and Daxx in apoptosis, we investigated whether Daxx and HIPK2 colocalize. Expression of GFP-HIPK2 revealed that a significant proportion of endogenous Daxx colocalizes with HIPK2 in NBs 12 h after transfection (Fig. 1A)Citation . Consistent with our previous findings (4) , GFP-HIPK2 colocalizes under these conditions with endogenous PML in PML-NBs (Fig. 1B)Citation , indicating that Daxx and HIPK2 colocalize in PML-NBs. Interestingly, after prolonged expression of HIPK2 (16 h after transfection) Daxx showed a slightly changed distribution in NBs and an increased nucleoplasmic staining pattern (Fig. 1C)Citation . Similar as very recently reported for hamster HIPK2/PKM (32) , expression of HIPK2 resulted in the disruption of PML-NBs (Fig. 1E)Citation , and the subsequent release of Daxx (Fig. 1D)Citation and PML (Fig. 1E)Citation 24 h after transfection. In contrast, kinase-deficient HIPK2K221A did not colocalize with Daxx and failed to release Daxx from the PML-NBs (Fig. 1F)Citation . Thus, our data indicate that wild-type HIPK2 is targeted to PML-NBs and leads to PML-NB disruption, which results in the mobilization of Daxx.



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  		Fig. 1. HIPK2 colocalizes with Daxx and releases it from PML-NBs. A, U2OS cells were transfected with GFP-HIPK2 (green), and the subcellular distribution of endogenous Daxx protein (red) was analyzed 12 h post-transfection after fixation by autofluorescence of GFP (HIPK2) or by indirect immunofluorescence staining using a rabbit anti-Daxx antibody (M-112) and an Alexa594-conjugated goat antirabbit secondary antibody. DNA was visualized by Draq5 staining. A representative confocal image is shown. B, cells were transfected as in A, and endogenous PML (red) was stained using a mouse anti-PML antibody (PG-M3) and an Alexa594-conjugated goat antirabbit secondary antibody. A representative confocal image is shown. C and D, cells were transfected as described in A, and endogenous Daxx (red) and GFP-HIPK2 (green) distribution was analyzed at the indicated time points (C, 16 h and D, 24 h after transfection). E, U2OS cells were analyzed 24 h after transfection for the distribution of the exogenously expressed GFP-HIPK2 (green) and the endogenous Daxx protein (red) as described in A. F, U2OS were analyzed 24 h after transfection for ectopically expressed kinase deficient GFP-HIPK2K221A (green) and endogenous Daxx protein (red) as described in A.

 
In Vitro and in Vivo Interaction of HIPK2 and Daxx.
To investigate whether HIPK2 and Daxx interact, we performed GST-pulldown assays using bacterially expressed GST-fusion proteins and in vitro translated 35S-labeled proteins. Daxx as well as a COOH-terminal truncation of Daxx (aa 1–400) specifically interacted with COOH-terminal truncated GST-HIPK2{Delta}C (amino acids 1–520; Fig. 2ACitation ). No binding of Daxx to recombinant NH2-terminal truncated HIPK2 (GST-HIPK2{Delta}N; amino acids 551-1191) was observed (data not shown). In addition, using various recombinant GST-Daxx proteins we mapped a strong interaction between HIPK2 and the NH2-terminal 188 amino acids of Daxx (Fig. 2B)Citation . Long exposures of the gel also revealed a weak binding of HIPK2 to COOH-terminus of Daxx (GST-Daxx 601–740; data not shown).



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  		Fig. 2. In vitro and in vivo interaction of HIPK2 and Daxx. A, GST-pulldowns were performed using GST-HIPK2{Delta}C (GST-HIPK2 1–520) fusion protein and the different in vitro translated 35S-labeled Daxx proteins indicated. A representative autoradiography (top part) and the Coomassie Brilliant Blue (CBB) staining of the GST-fusions (bottom part) is shown. B, left: schematic representation of the GST-Daxx fusion proteins. CC, coiled-coil domain, AD, acidic domain. Right: GST-pulldowns were performed with GST-Daxx proteins and in vitro translated 35S-labeled HIPK2 proteins indicated. A representative autoradiography (top part) and the Coomassie Brilliant Blue (CBB) staining of the GST-fusions (bottom part) is shown. C, the different Flag-tagged HIPK2 proteins were immunoprecipitated from lysates from 293T cells transfected with the indicated expression vectors, and Daxx was detected by immunoblotting with anti-Daxx antibody (H-7). The interaction domains of Daxx and HIPK2 are schematically displayed in the bottom part. Molecular weight (kDa) protein markers are indicated. D, GFP-HIPK2 was immunoprecipitated from lysates from 293T cells transfected with the indicated expression vectors with anti-GFP polyclonal antibodies followed by detection of Flag-Daxx by immunoblotting with anti-Flag antibodies. Ten percent of the cell lysates used for immunoprecipitation were analyzed by immunoblotting with anti-Flag and anti-GFP antibodies as indicated. E, lysates from in vivo cross-linked U2OS cells were subjected to immunoprecipitation with control rabbit IgG antibodies or affinity purified rabbit anti-HIPK2 antibodies. Proteins were detected by immunoblotting with anti-HIPK2 and anti-Daxx (M-112) antibodies.

 
To test whether HIPK2 and Daxx also interact in vivo, we expressed Daxx together with various Flag-tagged HIPK2 proteins in 293T cells, immunoprecipitated the Flag-HIPK2 proteins from the lysates, and analyzed the precipitates by immunoblotting with anti-Daxx antibodies. Daxx specifically coimmunoprecipitated with HIPK2, whereas kinase-deficient HIPK2K221A (33) , although expressed to a higher level than HIPK2, reproducibly bound less efficiently, suggesting a preferential interaction of enzymatically active HIPK2 with Daxx (Fig. 2CCitation , left panel). In accordance with our in vitro mapping results, we mapped the in vivo interaction domain to the HIPK2 kinase domain (aa 189–520; Fig. 2CCitation , right panel). In addition, GFP-HIPK2 reciprocally immunoprecipitated with Flag-tagged Daxx and Flag-Daxx 1–400 from 293T cells (Fig. 2D)Citation .

Because the interaction of HIPK2 and Daxx appeared to be of transient nature (concluded from the finding that endogenous Daxx is released from PML-NBs by HIPK2 expression 24 h after transfection, whereas HIPK2 remains in NBs; Fig. 1DCitation ) we used an accepted in vivo cross-linking approach (34) to study the interaction of endogenous HIPK2 and endogenous Daxx proteins in U2OS cells. A fraction of Daxx specifically coprecipitated with HIPK2 (Fig. 2E)Citation , demonstrating that HIPK2 associates with Daxx under physiological conditions.

HIPK2 Regulates Daxx Phosphorylation.
Because HIPK2 binds Daxx via its kinase domain, we wondered whether HIPK2 phosphorylates Daxx. Ectopic expression of Flag-Daxx and Flag-HIPK2 in 293 cells followed by immunoprecipitation and in vitro kinase assays revealed a robust Daxx phosphorylation in the presence of catalytically active HIPK2 (Fig. 3A)Citation . In contrast, only background phosphorylation was detectable when kinase-deficient HIPK2K221A was coexpressed (Fig. 3A)Citation . To test whether HIPK2 is able to induce Daxx phosphorylation in vivo, we performed in vivo [32P]orthophosphate labeling of 293 cells expressing untagged Daxx together with Flag-HIPK2 or Flag-HIPK2K221A. Daxx was immunoprecipitated, and the immunecomplexes were analyzed by SDS-PAGE and autoradiography (Fig. 3B)Citation . Strong in vivo phosphorylation of Daxx mediated by HIPK2 expression was observed. Consistent with these findings, Daxx is a phosphoprotein (14) , and murine Daxx was reported recently to be phosphorylated by ectopic expression of murine HIPK1 at Ser669 (35) . However, using various bacterially expressed GST-Daxx fusion proteins no evidence for direct Daxx phosphorylation by HIPK2 was obtained (data not shown). Provided the GST-Daxx fusion proteins mimicked the in vivo conformation of Daxx, our data suggest that HIPK2 indirectly induces Daxx phosphorylation.



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  		Fig. 3. HIPK2 expression induces Daxx phosphorylation. A, 293 cells were transfected with Flag-Daxx and either Flag-HIPK2 or kinase-deficient Flag-HIPK2K221A as indicated. Lysates were immunoprecipitated with anti-Flag antibodies, followed by in vitro kinase assays (KA; top panel). Expression levels of Flag-Daxx were examined by immunoblotting of the proteins contained in the cell lysates with anti-Flag antibodies (bottom panel). B, 293 cells were transfected with Daxx and either Flag-HIPK2 or Flag-HIPK2K221A. Thirty h later cells were labeled in vivo with [32P]orthophosphate. Daxx was immunoprecipitated using anti-Daxx (M-112) antibodies, and immunoprecipitates were analyzed by SDS-PAGE and autoradiography (KA; top panel). Expression levels of Daxx were examined by immunoblotting 10% of the cell lysates with anti-Daxx antibodies (H-7; bottom panel).

 
HIPK2 Activates the JNK Signaling Pathway.
Daxx has been reported previously to activate the JNK signaling pathway (10 , 11 , 23) . To test whether HIPK2 might be involved in JNK activation, we expressed HIPK2 or kinase-deficient HIPK2K221A together with HA-JNK1 in 293 cells. Immunecomplex kinase assays revealed a pronounced JNK activation by HIPK2, in contrast with HIPK2K221A (Fig. 4A)Citation . To obtain additional evidence for the stimulatory effect of HIPK2 on JNK, we tested for activation of serum response factor by using a SRE-containing luciferase reporter construct. JNK phosphorylates ternary complex factor and thereby activates the SRE (36) . HIPK2 strongly activated the SRE-luciferase reporter in a kinase-dependent manner (Fig. 4B)Citation . Next we tested whether the stimulatory effect of HIPK2 is specific for JNK, or whether it also effects other MAPKs. To this end, we used phosphospecific antibodies recognizing the activated, phosphorylated forms of the MAPK p38 or Erk1/2. HIPK2 failed to activate p38 (Fig. 4C)Citation or Erk1/2 (Fig. 4D)Citation . Furthermore, it also failed to activate a nuclear factor {kappa}B-dependent luciferase reporter plasmid (Fig. 4E)Citation . Taken together, these results indicate that HIPK2 can activate the JNK pathway.



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  		Fig. 4. HIPK2 activates the JNK signal transduction pathway. A, 293 cells were transfected with pcDNA3, Flag-HIPK2, Flag-HIPK2K221A, or Flag-TRAF2 together with HA-JNK1 as indicated. In vitro kinase assays (KA) with precipitated HA-JNK1 were performed in the presence of [{gamma}-32P]ATP and GST-cJun (5–89) as substrate (top panel). Ten percent of the cell lysate was analyzed by immunoblotting for expression of the Flag-tagged constructs (middle panel) and HA-JNK1 (bottom panel). B, 293 cells were transfected with 2 x SRE luciferase reporter plasmid and the indicated HIPK2 constructs. Mean values of four independent experiments are shown; bars, ±SD. C, 293 cells were transfected with the expression vectors indicated along with 1 µg Flag-p38. Equal protein amounts were analyzed by immunoblotting with anti-Flag, anti-HA, anti-p38, or anti-phospho-specific p38 antibodies as indicated. D, 293 cells were transfected with the expression constructs indicated. Lysates were analyzed by immunoblotting with anti-phospho-Erk, anti-Erk, anti-HA, and anti-Flag antibodies as indicated. E, HIPK2 and MEKK1{Delta} were expressed in 293 cells and tested for the activation of a cotransfected 3 x nuclear factor {kappa}B luciferase reporter gene. Luciferase activity was determined 24 h later. Mean values of four independent experiments are shown; bars, ±SD.

 
HIPK2 Activates JNK via a Pathway Using Daxx, MKK4, and MKK7.
JNK is activated through direct phosphorylation by two upstream MAPK kinases, MKK4/SEK1 (37) and MKK7 (38) . To examine the signaling pathway of HIPK2 toward JNK we performed in vitro JNK kinase assays by expressing HIPK2 and JNK1 together with trans-DN versions of MKK4 (Fig. 5A)Citation and MKK7 in 293 cells (Fig. 5B)Citation . Expression of either MKK4-DN or MKK7-DN led to a significant reduction of HIPK2-induced JNK activation, suggesting that HIPK2 uses both proteins to signal JNK activation. In contrast, a trans-DN version of the MEKK MEKK1 did not interfere with HIPK2-induced JNK activation (Fig. 5C)Citation .



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  		Fig. 5. HIPK2 activates JNK via Daxx, MKK4, and MKK7. A, 293 cells were transfected with pcDNA3, Flag-HIPK2, and increasing amounts of Flag-MKK7DN expression vector along with HA-JNK1 at the indicated combinations. In vitro kinase activity (KA) of precipitated HA-JNK1 was determined using GST-cJun (5–89) in presence of [{gamma}-32P]ATP (top panel). Ten percent of the cell lysate was analyzed by Western blotting (WB) for expression of the Flag-tagged constructs (middle panels) and HA-JNK1, respectively (bottom panel). 293 cells were transfected with Flag-HIPK2, HA-JNK1, and increasing amounts of B, Flag-MKK4DN, or C, HA-MEKK1{Delta}DN, respectively, and in vitro JNK activity (KA) was determined as described in A (top panel). Cell lysate was analyzed by Western blotting as described in A (middle and bottom panels). D, 293 cells were transfected with pcDNA3, Flag-HIPK2, and Flag-Daxx along with HA-JNK1 at the indicated combinations followed by analysis of in vitro JNK kinase activity (KA) as described in A (top panel). Ten percent of the cell lysate was analyzed by Western blotting (WB) for expression of the Flag-tagged constructs and Daxx (middle panels) and HA-JNK1, respectively (bottom panel). E, 293 cells were transfected as indicated with pcDNA3, Flag-HIPK2, or Flag-DaxxC together with HA-JNK1, followed by analysis of JNK activity (KA) as described in A.

 
Because HIPK2 and Daxx both activate the JNK signaling cascade, we next addressed the question of whether both proteins cooperate in JNK activation. To this end, 293 cells were transfected either with Daxx and HIPK2 expression constructs alone or in combination, and JNK activation was determined by in vitro kinase assays. Cotransfection of HIPK2 and Daxx increased HIPK2-mediated JNK activation (Fig. 5D)Citation , indicating that Daxx can cooperate with HIPK2 to activate JNK. For unknown reasons we did not observe JNK activation by expressing Daxx alone. DaxxC, a deletion mutant of Daxx comprising the COOH-terminus of Daxx, which interferes with CD95 and TGF-ß-induced JNK activation (10 , 23) and lacks the interaction domain with HIPK2, inhibited HIPK2-elicited JNK activation (Fig. 5E)Citation .

HIPK2 and Daxx Cooperate for TGF-ß-Induced Cell Death in Human Hepatoma Cells.
Next we analyzed whether HIPK2, similar to Daxx (23) , can regulate TGF-ß-induced cell death. To exclude any contribution of the known activating effect of HIPK2 on p53 (4 , 5) we used p53-negative Hep3B hepatocellular carcinoma cells, which are sensitive to TGF-ß-induced apoptosis (39 , 40) . Overexpression of Daxx or HIPK2 led to an increase in TGF-ß-induced apoptosis (Fig. 6)Citation . In contrast, expression of kinase-deficient HIPK2 failed to increase TGF-ß-induced cell death (Fig. 6)Citation , indicating that this effect is mediated by the kinase function. Coexpression of HIPK2 and Daxx resulted in potentiation of TGF-ß-induced cell death, whereas the combination of Daxx and kinase-deficient HIPK2 did not (Fig. 6)Citation . Taken together, our experiments indicate that HIPK2 sensitizes for TGF-ß-induced cell death, and this depends on its kinase-activity, which is essential for its JNK activating function.



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  		Fig. 6. HIPK2 and Daxx cooperate for TGF-ß-induced apoptosis in a JNK-dependent manner in human hepatoma cells. A, Hep3B cells were transfected with empty pcDNA3 vector or the indicated Flag-HIPK2 and Daxx constructs along with pEGFP-C1 vector as a marker for transfected cells and treated with 4 ng/ml TGF-ß. Twenty-four h later DAPI was added to the cell culture medium and 150 GFP-positive cells for each transfection were analyzed under a fluorescence microscope for apoptosis-associated chromatin condensation. GFP-expressing cells with condensed, apoptotic nuclei (compare top panel) were counted as apoptotic. Mean values of three independent experiments are shown; bars, ±SD.

 
HIPK2 Regulates TGF-ß-Induced JNK Activation and Apoptosis.
Next, we addressed the potential role of endogenous HIPK2 in TGF-ß-induced JNK activation and apoptosis in Hep3B cells. As expected, immunecomplex kinase assays with immunoprecipitated endogenous JNK1 and JNK2 from Hep3B cells revealed a time-dependent increase in TGF-ß-induced JNK activation with strongest induction 16 h after stimulation (Fig. 7ACitation ; data not shown). To test whether this increase depends on HIPK2 expression, we knocked down endogenous HIPK2 in Hep3B cells by RNA interference (31) using two different HIPK2-specific siRNAs as well as a luciferase control siRNA (Fig. 7B)Citation . Because knock down of endogenous HIPK2 was most efficient 24 h after transfection, TGF-ß stimulation was started at this time point, and endogenous JNK activation was analyzed 16 h thereafter. Compared with control-treated cells, TGF-ß-induced JNK activation was impaired after knock down of HIPK2 (Fig. 7C)Citation . Consistent with the reduced JNK activation, down-regulation of endogenous HIPK2 significantly protected Hep3B cells from TGF-ß-mediated apoptosis (Fig. 7D)Citation .



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  		Fig. 7. Endogenous HIPK2 regulates TGF-ß-induced JNK activation and apoptosis in human hepatoma cells. A, Hep3B cells were incubated for 6 h in DMEM/0.1% FCS and subsequently treated with 4 ng/ml TGF-ß as indicated. After the indicated time points, JNK activation was monitored by immunoprecipitation of endogenous JNK1/JNK2 and subsequently in vitro JNK assays (KA) were performed. B, Hep3B cells were transfected with control luciferase siRNA or HIPK2-specific siRNAs. Total cell lysates were analyzed for endogenous HIPK2 and ß-actin protein expression. C, Hep3B cells were transfected with the indicated siRNAs and incubated in DMEM/0.1% FCS. Twenty-four h after transfection cells were stimulated with 4 ng/ml TGF-ß and JNK activation (top panel) was monitored 16 h after stimulation as in A. Ten percent of the cell lysates were analyzed by immunoblotting with anti-JNK antibodies (bottom panel). D, Hep3B cells were treated with HIPK2-specific siRNAs or control siRNA and maintained in DMEM/0.1% FCS for 24 h before administration of 4 ng/ml TGF-ß. Apoptosis was determined 30 h later using trypan blue exclusion. Mean values of three independent experiments are shown; bars, ±SDs.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we provide evidence for a physical and functional interaction of HIPK2 and Daxx. We show that HIPK2 regulates Daxx phosphorylation and that enforced expression of HIPK2 leads to PML-NB disintegration, and thereby releases Daxx from PML-NBs into the nucleoplasm. Furthermore, we demonstrated that HIPK2 and Daxx cooperatively activate the JNK signaling pathway, and that HIPK2 regulates TGF-ß-induced JNK activation and apoptosis in p53-deficient hepatoma cells.

The Role of HIPK2 and Daxx in Apoptosis.
Daxx is a multifunctional protein mediating JNK activation and apoptosis in CD95 (Fas/APO-1; Refs. 10 , 11 ) and TGF-ß-induced apoptosis (23) . TGF-ß exerts its growth-inhibiting function basically by two mechanisms. It either leads to cell cycle arrest (26 , 41) or triggers apoptosis in specific cell types including hepatocytes and B cells (42 , 43) . Daxx was shown to be an essential adaptor protein to signal TGF-ß-induced JNK activation and apoptosis (23) . Although we did not find elevated HIPK2 kinase activity after TGF-ß treatment in Hep3B cells (data not shown), the impaired TGF-ß-induced JNK activation and apoptosis in response to siRNA-mediated knock down of endogenous HIPK2, as well as the increased sensitivity toward TGF-ß-induced cell death after HIPK2 overexpression, strongly suggests a role of HIPK2 in the regulation of this signaling pathway. We speculate that in addition, not yet identified HIPK2 activating signals contribute to sensitization to TGF-ß-induced apoptosis, or that HIPK2 fulfils an adaptor protein function similar to that of the interleukin-1 receptor-activated kinases in Toll-like/interleukin 1 receptor family signaling (44) . Because coexpression of Daxx and HIPK2 increases HIPK2-induced JNK activation, and furthermore DaxxC partially inhibits HIPK2-induced JNK activation, we suggest that both proteins signal via an overlapping pathway. However, the partial inhibition of HIPK2-induced JNK activation by DaxxC suggests that HIPK2 in addition signals to JNK via other proteins. Interestingly, ASK1 is activated by its association with Daxx and induces JNK activation after CD95 (Fas/APO-1) ligation, and moreover ASK1 recruits Daxx to the cytoplasmic compartment where both elicit a death-inducing signaling pathway (11 , 20 , 21) . The contribution of HIPK2 to TGF-ß-induced apoptosis may rely on the mobilization of Daxx from PML-NBs, and subsequently ASK1 may shuttle Daxx to the cytoplasm where it acts in TGF-ß-induced signal transduction.

The Function of HIPK2 and Daxx in Transcriptional Regulation.
Daxx functions as a transcriptional repressor, and its repressor function is released through its recruitment to PML-NBs (16 , 17) . How proteins once recruited to PML-NBs, such as Daxx, are again remobilized from PML-NBs remained thus far unclear. Our finding that HIPK2 disintegrates PML-NBs suggests a possible mechanism of how cells obtain access to factors once recruited to PML-NBs and target them to their cellular destination at the chromatin or the cytoplasmic compartment, where they regulate gene expression and apoptosis. Similar to HIPK2 (4) , Daxx can bind and cooperate with CREB-binding protein (CBP) for transcriptional activation (18) , demonstrating that Daxx, dependent on its molecular context, functions as a transcriptional corepressor or coactivator. Importantly, CBP also acts as a cofactor for Smad proteins in TGF-ß signaling (45 , 46) . Therefore, it will be interesting to determine in the future whether HIPK2 can also modulate Smad function. Interestingly, Daxx expression is paralleled by Bcl-2 down-regulation and apoptosis induction upon IFN treatment, suggesting that Daxx-mediated gene regulation can directly down-regulate the expression of antiapoptotic factors (47) . Thus, HIPK2 may also modulate TGF-ß-induced apoptosis by repressing antiapoptotic regulators such as Bcl-2 in concert with Daxx.

HIPKs and Their Role in Daxx Phosphorylation.
Daxx is a highly phosphorylated protein (14) , and a very recent report showed that murine HIPK1, a close relative of human HIPK2, interacts with Daxx and that its ectopic expression triggers Daxx phosphorylation at serine 669 (35) . Similarly, we found that human HIPK2 induces Daxx phosphorylation on overexpression in 293T cells. However, we failed to phosphorylate various GST-Daxx fusion proteins (which altogether span the entire amino acid sequence of Daxx) by HIPK2 (data not shown), suggesting that the phosphorylation is indirect or requires post-translational modifications absent in bacterially expressed fusion proteins. Thus, future studies are needed to clarify the role of HIPKs in Daxx phosphorylation. Interestingly, expression of murine HIPK1 does not disintegrate PML-NBs but interferes with the interaction of Daxx with PML (35) , indicating that human HIPK2 and also hamster HIPK2/PKM (32) use a different molecular mechanism to release Daxx from PML-NBs.

PML-NBs: Regulatory Domains for Daxx Function.
Recent reports indicate that PML-NBs act as regulatory sites for Daxx function by controlling its bioavailability, and, thus, influencing Daxx-dependent effector pathways (15 , 48) . On the basis of these and our data presented here, we propose a model that HIPK2 is recruited to PML-NBs, interacts with Daxx, and releases it by disintegrating PML-NBs. Thus, HIPK2 targets Daxx to its subcellular sites of function by releasing it from PML-NBs, and sequestering it to nucleoplasmic and cytoplasmic compartments, where Daxx can participate in transcriptional regulation and TGF-ß-induced apoptosis signaling, respectively.


   ACKNOWLEDGMENTS
 
We thank X. Yang, David Baltimore, A. D. Sharrocks, T. Heise, T. Sternsdorf, R. J. Davis, E. Nishida, T. Maniatis, K. Herzer, and P. G. Pelicci for generously providing reagents and protocols.


   FOOTNOTES
 
Grant support: Fonds der chemischen Industrie, EU project (QLK3-CT-2000-00463) sponsored by the Schweizerisches Bundesamt für Bildung und Wissenschaft, Schweizerischer Nationalfonds, Association for International Cancer Research, the "Stiftung zur Förderung der wissenschaftlichen Forschung an der Universität Bern," the Roche Foundation, the "Stiftung zur Bekämpfung Neuroviraler Erkrankungen," the Deutsche Krebshilfe (Wi2-10-1624) and the Deutsche Forschungsgemeinschaft (DFG; HO2438/2-1, WI664/6-2). The Heinrich-Pette-Institut is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: To whom requests for reprints should be addressed, at Thomas Hofmann, Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistr. 52, Hamburg 20251, Germany. Phone: 49-40-48051322; Fax: 49-40-48051222; E-mail: thomas.hofmann{at}hpi.uni-hamburg.de

3 The abbreviations used are: HIPK, homeodomain-interacting protein kinase; DAPI, 4',6-diamidino-2-phenylindole; MEKK, mitogen-activated protein kinase kinase kinase; PML, promyelocytic leukemia; NB, nuclear body; GST, glutathione S-transferase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; TGF, transforming growth factor; APL, acute promyelocytic leukemia; ASK1, apoptotic signal regulating kinase 1; Erk, extracellular signal-regulated kinase; aa, amino acid; SRE, serum response element; DN, dominant negative. Back

Received 6/ 2/03. Revised 8/16/03. Accepted 9/19/03.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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