Homeodomain-interacting Protein Kinase 2 Overexpression Research Articles

MiR-664a-5p promotes experimental membranous nephropathy progression through HIPK2/Calpain1/GSα-mediated autophagy inhibition.

We previously found that miR-664a-5p is specifically expressed in urinary exosomes of idiopathic membranous nephropathy (IMN) patients. Homeodomain-interacting protein kinase 2 (HIPK2), a nuclear serine/threonine kinase, plays an important role in nephropathy. But the function of these factors and their connection in MN are unclear. To investigate the function and mechanism of miR-664a-5p in MN, the miR-664a-5p expression in HK-2 cells, exosomes, podocytes and renal tissues were studied, as well as cell growth and apoptosis of these cells, the binding of miR-664a-5p to HIPK2 mRNA, the levels of relative proteins and autophagy. The MN progression in MN mice model was also studied. Albumin increased the miR-664a-5p content and apoptosis of HK-2 cells, which was blocked by miR-664a-5p antagomir. miR-664a-5p bound to the 3' UTR of HIPK2 mRNA, resulting in the up-regulation of Calpain1, GSα shear and the inhibition of autophagy level. Autophagy inhibitor CQ blocked the protective effect of miR-664a-5p antagomir, HIPK2 overexpression, Calpain inhibitor SJA6017 on albumin-mediated injury. MiR-664a-5p from albumin-treated HK-2 cells could be horizontally transported to podocytes through exosomes. Exosomes from albumin-treated HK-2 cells promoted progression of MN mice, AAV-Anti-miR-664-5p (mouse homology miRNA) could improve them. Albumin increases the miR-664a-5p level and causes changes of HIPK2/Calpain1/GSα pathway, which leads to autophagy inhibition and apoptosis up-regulation of renal tubular epithelial cells. miR-664a-5p can horizontally enter podocytes through exosomes resulting in podocytes injury. Targeted inhibition of miR-664a-5p can reduce the apoptosis of renal tubule cells and podocytes, and may improve the MN progression.

Homeodomain-interacting protein kinase 2 regulates NLRP3 inflammasome activation through endoplasmic reticulum stress in septic liver injury.

Septic liver injury is a major burden for the clinical management of sepsis. Hepatocyte cell death plays a crucial pathophysiological role in sepsis. A recent study proposed that NLRP3 inflammasome-mediated pyroptosis participates in septic liver injury. Therefore, investigating the mechanism controlling this process may help manage sepsis. We investigated the role of homeodomain-interacting protein kinase 2 (HIPK2) in regulating the NLRP3 inflammasome in vivo using mouse models and in vitro in primary hepatocytes. HIPK2 could improve liver injury and survival in a mouse model of sepsis. Overexpression of HIPK2 could suppress NLRP3 and caspase-1-p20 expression, while HIPK2 knockdown led to higher levels of these two molecules. Importantly, HIPK2 could suppress endoplasmic reticulum (ER) stress. Pharmacologically inhibiting ER stress could abolish activation of the NLRP3 inflammasome in hepatocytes with HIPK2 knockdown. HIPK2 can regulate ER stress and NLRP3 inflammasome activation in the liver during sepsis, and HIPK2-mediated suppression of ER stress participates in regulating NLRP3 inflammasome activation. The present study highlights the role of HIPK2 in regulating the inflammasome in septic liver injury, which may serve as a target for managing sepsis.

Homeodomain Interacting Protein Kinase 2-Modified Rat Spinal Astrocytes Affect Neurofunctional Recovery After Spinal Cord Injury.

Spinal cord injury (SCI) is regarded as an acute neurological disorder, and astrocytes play a role in the progression of SCI. Herein, we investigated the roles of homeodomain-interacting protein kinase 2 (HIPK2)- modified rat spinal astrocytes in neurofunctional recovery after SCI. Rat spinal astrocytes were cultured, isolated, and then identified through microscopic observation and immunofluorescence staining. Astrocytes were infected with the adenovirus vector overexpressing HIPK2 for modification, and proliferation and apoptosis of astrocytes were examined using Cell Counting Kit-8 method and flow cytometry. SCI rat models were established and treated with astrocytes or HIPK2-modified astrocytes. Subsequently, rat motor ability was analyzed via the Basso-Beattie-Bresnahan (BBB) scoring and inclined-plane test, and the damage to spinal cord tissues and neuronal survival were observed via Hematoxylin-eosin staining and Nissl staining. The levels of HIPK2, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and nuclear factor erythroid 2- related transcription factor 2 (Nrf2)/antioxidant response element (ARE) pathway-related proteins were detected. Rat spinal astrocytes were harvested successfully. HIPK2 overexpression accelerated the proliferation and repressed the apoptosis of rat spinal astrocytes. Rat spinal astrocytes treatment increased BBB points and the maximum angle at which SCI rats remained stable, ameliorated damage to spinal cord tissues, increased the number of neurons, and attenuated neural damage and inflammation, while the treatment of HIPK2-modified rat spinal astrocytes imparted more pronounced effects to the neurofunctional recovery of SCI rats. Meanwhile, HIPK2-modified rat spinal astrocytes further activated the Nrf2/ARE pathway. HIPK2-modified rat spinal astrocytes facilitated neurofunctional recovery and activated the Nrf2/ARE pathway after SCI.

Overexpression of HIPK2 removes the transrepression of proapoptotic genes mediated by the CtBP1-p300-FOXO3a complex and increases the chemosensitivity in osteosarcoma cells.

Decreased expression of proapoptotic genes can lead to the chemoresistenance in cancer therapy. Carboxyl-terminal binding protein 1 (CtBP1), a transcriptional corepressor with multiple oncogenic effects, has been previously identified to suppress the expression of two proapoptotic genes [BAX (BCL2 associated X) and BIM (Bcl-2 interacting mediator of cell death)] by assembling a complex with the Forkhead box O3 (FOXO3a) transcription factor and the p300 histone acetyltransferase. However, the upstream regulatory signaling of the CtBP1-p300-FOXO3a complex is obscure, and the effects of changing this signaling on chemosensitivity in osteosarcoma are unknown. Herein, we discovered that the downregulation of HIPK2 (Homeodomain-interacting protein kinase 2) was essential for the function of the CtBP1-p300-FOXO3a complex. Downregulation of HIPK2 prevented the phosphorylation and subsequent degradation of CtBP1, thereby allowing the assembly of the CtBP1-p300-FOXO3a complex and suppression of the expression of proapoptotic genes, such as BAX, BIM, BIK (Bcl-2 interacting killer) and NOXA/PMAIP1 (Phorbol-12-myristate-13-acetate-induced protein 1). Overexpression of HIPK2 promoted the phosphorylation of CtBP1 and the degradation of CtBP1 by proteasomes, thereby preventing the formation of the CtBP1-p300-FOXO3a complex. The abolition of CtBP1 transrepression increased the expression of proapoptotic genes to induce apoptosis and increase chemosensitivity in osteosarcoma cells. Taken together, our in vitro and in vivo results revealed that overexpression of HIPK2 could remove the CtBP1-mediated transrepression of proapoptotic genes, indicating a new therapeutic option for the treatment of osteosarcoma.

Tubular HIPK2 is a key contributor to renal fibrosis.

We previously used global Hipk2-null mice in various models of kidney disease to demonstrate the central role of homeodomain-interacting protein kinase 2 (HIPK2) in renal fibrosis development. However, renal tubular epithelial cell–specific (RTEC-specific) HIPK2 function in renal fibrogenesis has yet to be determined. Here, we show that modulation of tubular HIPK2 expression and activity affects renal fibrosis development in vivo. The loss of HIPK2 expression in RTECs resulted in a marked diminution of renal fibrosis in unilateral ureteral obstruction (UUO) mouse models and HIV-associated nephropathy (HIVAN) mouse models, which was associated with the reduction of Smad3 activation and downstream expression of profibrotic markers. Conversely, WT HIPK2 overexpression in RTECs accentuated the extent of renal fibrosis in the setting of UUO, HIVAN, and folic acid–induced nephropathy in mice. Notably, kinase-dead HIPK2 mutant overexpression or administration of BT173, an allosteric inhibitor of HIPK2-Smad3 interaction, markedly attenuated the renal fibrosis in these mouse models of kidney disease, indicating that HIPK2 requires both the kinase activity and its interaction with Smad3 to promote TGF-β–mediated renal fibrosis. Together, these results establish an important RTEC-specific role of HIPK2 in kidney fibrosis and further substantiate the inhibition of HIPK2 as a therapeutic approach against renal fibrosis.

HIPK2 suppresses tumor growth and progression of hepatocellular carcinoma through promoting the degradation of HIF-1α.

Aberrant angiogenesis of hepatocellular carcinoma (HCC) leads to tumor growth and local or distant metastasis. Uncovering the underlying mechanisms for the neoangiogenesis of HCC can provide novel potential therapeutic targets in the clinic. Here, we reported that serine/threonine homeodomain-interacting protein kinase 2 (HIPK2) was frequently downregulated in HCC tissues compared with the adjacent normal tissues, and patients with lower HIPK2 protein expression were associated with worse overall survival. Both in vitro and in vivo, HIPK2 inhibited the migration of HCC cells, as well as tumor growth and metastasis in xenograft and orthotopic syngeneic HCC mouse models. Furthermore, HIPK2 inhibited the angiogenesis in HCC tumors. Under the hypoxic condition, HIPK2 knockdown enhanced the angiogenesis and the key regulator, HIF-1α signaling pathway; however, HIPK2 overexpression downregulated the tumoral angiogenesis and HIF-1α signaling. In HCC cells, HIPK2 could directly bind to HIF-1α and stimulate the ubiquitination of HIF-1α for proteasomal degradation. HIF-1α knockout partially rescued the promoting effect of HIPK2 depletion on angiogenesis and tumor growth. In conclusion, the downregulation of HIPK2 could enhance the angiogenesis in HCC through inducing the HIF-1α pathway, and further contribute to tumor growth and metastasis, which may provide a novel therapeutic strategy for HCC.

HIPK2 overexpression relieves hypoxia/reoxygenation-induced apoptosis and oxidative damage of cardiomyocytes through enhancement of the Nrf2/ARE signaling pathway

Homeodomain interacting protein kinase-2 (HIPK2) has emerged as a crucial stress-responsive kinase that plays a critical role in regulating cell survival and apoptosis. However, whether HIPK2 participates in regulating cardiomyocyte survival during myocardial ischemia/reperfusion injury remains unclear. Here, we investigated the regulatory effect of HIPK2 on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its potential underlying molecular mechanism. We found that HIPK2 expression was induced in response to H/R exposure. HIPK2 depletion by small interfering RNA (siRNA)-mediated gene silencing significantly decreased the viability and exacerbated H/R-induced apoptosis and reactive oxygen species (ROS) production in cardiomyocytes. Comparatively, HIPK2 overexpression effectively rescued H/R-impaired viability and repressed H/R-induced apoptosis and ROS production in cardiomyocytes. HIPK2 overexpression significantly increased the nuclear expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and enhanced Nrf2-mediated transcriptional activity. Moreover, HIPK2 overexpression significantly increased the transcription of Nrf2/ARE target genes. Additionally, Nrf2 inhibition partially reversed the HIPK2-mediated protective effect. Overall, these results demonstrate that HIPK2 overexpression protects cardiomyocytes from H/R-induced injury by enhancing Nrf2/ARE antioxidant signaling, data that suggest HIPK2 is a potential target for cardioprotection.

Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling.

Cardiac kinases play a critical role in the development of heart failure, and represent potential tractable therapeutic targets. However, only a very small fraction of the cardiac kinome has been investigated. To identify novel cardiac kinases involved in heart failure, we used an integrated transcriptomics and bioinformatics analysis and identified Homeodomain-Interacting Protein Kinase 2 (HIPK2) as a novel candidate kinase. The role of HIPK2 in cardiac biology is unknown. We used the Expression2Kinase algorithm for the screening of kinase targets. To determine the role of HIPK2 in the heart, we generated cardiomyocyte (CM)-specific HIPK2 knockout and heterozygous mice. Heart function was examined by echocardiography, and related cellular and molecular mechanisms were examined. Adeno-associated virus serotype 9 carrying cardiac-specific constitutively active MEK1 (TnT-MEK1-CA) was administrated to rescue cardiac dysfunction in CM-HIPK2 knockout mice. To our knowledge, this is the first study to define the role of HIPK2 in cardiac biology. Using multiple HIPK2 loss-of-function mouse models, we demonstrated that reduction of HIPK2 in CMs leads to cardiac dysfunction, suggesting a causal role in heart failure. It is important to note that cardiac dysfunction in HIPK2 knockout mice developed with advancing age, but not during development. In addition, CM-HIPK2 knockout mice and CM-HIPK2 heterozygous mice exhibited a gene dose-response relationship of CM-HIPK2 on heart function. HIPK2 expression in the heart was significantly reduced in human end-stage ischemic cardiomyopathy in comparison to nonfailing myocardium, suggesting a clinical relevance of HIPK2 in cardiac biology. In vitro studies with neonatal rat ventricular CMscorroborated the in vivo findings. Specifically, adenovirus-mediated overexpression of HIPK2 suppressed the expression of heart failure markers, NPPA and NPPB, at basal condition and abolished phenylephrine-induced pathological gene expression. An array of mechanistic studies revealed impaired extracellular signal-regulated kinase 1/2 signaling in HIPK2-deficient hearts. An in vivo rescue experiment with adeno-associated virus serotype 9 TnT-MEK1-CA nearly abolished the detrimental phenotype of knockout mice, suggesting that impaired extracellular signal-regulated kinase signaling mediated apoptosis as the key factor driving the detrimental phenotype in CM-HIPK2 knockout mice hearts. Taken together, these findings suggest that CM-HIPK2 is required to maintain normal cardiac function via extracellular signal-regulated kinase signaling.

EGFR drives the progression of AKI to CKD through HIPK2 overexpression.

The molecular mechanism underlying the transition of acute kidney injury (AKI) to chronic kidney disease (CKD) induced by vancomycin (VAN) remains largely unknown.Methods: The mice model of VAN drives AKI to CKD was developed to investigate the role and molecular mechanism of epidermal growth factor receptor (EGFR). The EGF receptor mutant (Wa-2) mice and gefitinib were used to inactivation of EGFR. The homeodomain interacting protein kinase 2 (HIPK2) siRNA was applied to silence of HIPK2. Human proximal tubular epithelial cells (HK-2) were used to explore the molecular regulation methanism of EGFR. ChIp analysis was used to investigate if STAT3 interaction with the promoter of HIPK2.Results: A novel VAN-induced AKI mouse model was established for the first time. Moreover, the expression levels collagen I&IV, α-SMA, p-EGFR and the expression of HIPK2 proteins were upregulated in this model. Interestingly, AKI caused by VAN was markedly attenuated in waved-2 mice at the early stage, as evidenced by the suppression of renal dysfunction, renal cell apoptosis and caspase3 activation. In the latter stage, renal fibrosis and inflammation were significantly ameliorated in Wa-2 mice, accompanied by the downregulation of profibrotic molecules and F4/80. Besides, the expression levels of HIPK2 and p-STAT3 were suppressed in Wa-2 mice during VAN-induced transition of AKI to CKD. In addition, renal fibrosis and inflammation, profibrotic molecules, and EGFR/STAT3/HIPK2 signaling were ameliorated by gefitinib treatment after VAN-induced AKI. These results were consistent with the findings of Wa-2 mice. EGFR/STAT3 signaling mediated VAN-induced HIPK2 expression in HK-2 cells. ChIp analysis revealed that STAT3 directly bound to the promoter region of HIPK2. Finally, inhibition of HIPK2 attenuated the VAN drove the progression of AKI to CKD.Conclusion: These data suggest that EGFR plays an important role in VAN-driven progression of AKI to CKD.

Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

Sepsis is the leading cause of death in intensive care units worldwide. Autophagy has recently been shown to protect against sepsis-induced liver injury. Here, we investigated the roles of homeodomain-interacting protein kinase 2 (HIPK2) in the molecular mechanism of sepsis-induced liver injury. HIPK2 expression was reduced in sepsis-induced liver injury, and HIPK2 overexpression increased the survival rate and improved caecal ligation and puncture (CLP)-induced liver injury by reducing serum and liver aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) levels in mice with sepsis. HIPK2 overexpression significantly decreased CLP-induced release of inflammatory cytokines into the serum and attenuated oxidative stress-associated indicators in mice with CLP-induced liver injury, whereas HIPK2 knockdown produced the opposite results, suggesting that HIPK2 is a negative regulator of sepsis. Furthermore, HIPK2 overexpression inhibited lipopolysaccharide (LPS)-induced apoptosis of primary hepatocytes, increased the autophagic flux, and restored both autophagosome and autolysosome formation in the livers of CLP-induced mice by suppressing calpain signalling. Importantly, HIPK2 overexpression reduced the elevated cytosolic Ca2+ concentration in LPS-treated primary hepatocytes by interacting with calpain 1 and calmodulin. Finally, several anti-inflammatory drugs, including resveratrol, aspirin, vitamin E and ursolic acid, significantly increased the levels of the HIPK2 mRNA and protein by modulating promoter activity and the 3′-UTR stability of the HIPK2 gene. In conclusion, HIPK2 overexpression may improve sepsis-induced liver injury by restoring autophagy and thus might be a promising target for the clinical treatment of sepsis.

MicroRNA-222-3p associated with Helicobacter pylori targets HIPK2 to promote cell proliferation, invasion, and inhibits apoptosis in gastric cancer.

Gastric cancer ranks as the second leading cause of malignancy-related death worldwide, and always diagnosed at advanced stage. MicroRNA-222-3p (miR-222-3p) is aberrantly upregulated in various malignant tumors including gastric cancer, but its role and underlying molecular mechanisms in gastric cancer remain largely unknown. Helicobacter pylori (H. pylori) infection acts as a trigger in the development of gastric cancer, and increasing evidence suggests that H. pylori affects microRNA expression. In this study, gastric cancer tissue samples were divided into H. pylori positive group (+) and negative group (-). QRT-PCR showed that miR-222-3p was significantly upregulated in H. pylori (+) group compared with H. pylori (-) group, and luciferase reporter assays identified homeodomain-interacting protein kinase 2 (HIPK2) as a novel target of miR-222-3p in gastric cancer. Immunohistochemistry revealed that HIPK2 levels were decreased in H. pylori (+) group compared with H. pylori (-). After that, functional experiments indicated that miR-222-3p overexpression promoted the proliferation and invasion, while inhibiting apoptosis of SGC7901 gastric cancer cells, but miR-222-3p knockdown exhibited the opposite effects. Also, HIPK2 knockdown induced similar effects as miR-222-3p overexpression in SGC7901 cells. Nude mouse experiments further suggested that HIPK2 overexpression signally attenuated the enhancing effect of miR-222-3p overexpression on cell proliferation, indicating that the effect of miR-222-3p on gastric cancer progression depends on HIPK2, at least in part. Overall, our results demonstrated that miR-222-3p/HIPK2 signal pathway regulated gastric cancer cell proliferation, apoptosis, and invasion, provided a novel therapeutic target for the treatment of gastric cancer infected by H. pylori.

HIPK2 inhibits cell metastasis and improves chemosensitivity in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and lethal malignancies worldwide. At present, the underlying mechanisms of ESCC development and progression are poorly understood. Previous studies have demonstrated that homeodomain-interacting protein kinase-2 (HIPK2) serves an important role in cancer biology, particularly in proliferation and metastasis. However, the role of HIPK2 in ESCC cells remains unclear. In the current study, the expression of HIPK2 in ESCC specimens, adjacent non-cancerous tissues and cell lines was assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of HIPK2 on cell metastasis, epithelial-mesenchymal transition (EMT) and proliferation were studied using a Transwell assay, RT-qPCR and a Cell Counting Kit-8 assay, respectively. The results indicated that HIPK2 expression was downregulated in ESCC specimens and cell lines, and HIPK2 expression was associated with tumor invasion and lymph node metastasis. Functional studies demonstrated that HIPK2 overexpression inhibited cell metastasis and EMT. Furthermore, HIPK2 overexpression suppressed cell viability during cisplatin treatment. These results suggest that HIPK2 serves an important role in regulating metastasis and the chemosensitivity of ESCC cells, implicating the potential application of HIPK2 in ESCC therapy.

HIPK2 Overexpression and Its Prognostic Role in Human Papillomavirus-Positive Tonsillar Squamous Cell Carcinoma.

Tonsillar squamous cell carcinomas (TSCCs) are the most common human papillomavirus- (HPV-) associated oropharyngeal cancers with poor prognosis. Homeodomain-interacting protein kinase 2 (HIPK2) is a central regulator of p53, which participates in apoptosis during the DNA damage response. HIPK2 is involved in HPV-associated uterine cervical and cutaneous carcinogenesis through its binding of HPV E6, thereby preventing apoptosis and contributing to tumor progression. However, its clinical and prognostic significance in TSCC remains unclear. HIPK2 mRNA levels were analyzed in 20 normal tonsils and 20 TSCC specimens using real-time reverse transcription polymerase chain reaction. Immunohistochemistry of HIPK2 was performed in 79 resected specimens. HIPK2 was expressed in 57% of the TSCCs, and HIPK2 protein expression and HIPK2 mRNA levels were higher in TSCCs than in normal tonsils. HIPK2 overexpression was associated with poorly differentiated carcinoma and low alcohol consumption and was an independent prognostic factor for overall survival and disease-free survival (DFS) in TSCC and a negative independent prognostic factor for DFS in patients receiving postoperative radiotherapy. HIPK2 overexpression had a significant association with poorer DFS in HPV-positive TSCCs, but not in HPV-negative tumors. HIPK2 overexpression may be a potential prognostic marker for predicting prognoses and a high risk of recurrence, particularly in patients with HPV-positive TSCC.

Downregulation of HIPK2 increases resistance of bladder cancer cell to cisplatin by regulating Wip1.

Cisplatin-based combination chemotherapy regimen is a reasonable alternative to cystectomy in advanced/metastatic bladder cancer, but acquisition of cisplatin resistance is common in patients with bladder cancer. Previous studies showed that loss of homeodomain-interacting protein kinase-2 (HIPK2) contributes to cell proliferation and tumorigenesis. However, the role of HIPK2 in regulating chemoresistance of cancer cell is not fully understood. In the present study, we found that HIPK2 mRNA and protein levels are significantly decreased in cisplatin-resistant bladder cancer cell in vivo and in vitro. Downregulation of HIPK2 increases the cell viability in a dose- and time-dependent manner during cisplatin treatment, whereas overexpression of HIPK2 reduces the cell viability. HIPK2 overexpression partially overcomes cisplatin resistance in RT4-CisR cell. Furthermore, we showed that Wip1 (wild-type p53-induced phosphatase 1) expression is upregulated in RT4-CisR cell compared with RT4 cell, and HIPK2 negatively regulates Wip1 expression in bladder cancer cell. HIPK2 and Wip1 expression is also negatively correlated after cisplatin-based combination chemotherapy in vivo. Finally, we demonstrated that overexpression of HIPK2 sensitizes chemoresistant bladder cancer cell to cisplatin by regulating Wip1 expression.ConclusionsThese data suggest that HIPK2/Wip1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of bladder cancer.

HIPK2 phosphorylates ΔNp63α and promotes its degradation in response to DNA damage

Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging player in cell response to genotoxic agents that senses damage intensity and contributes to the cell's choice between cell cycle arrest and apoptosis. Phosphorylation of p53 at S46, an apoptosis-specific p53 posttranslational modification, is the most characterized HIPK2 function in response to lethal doses of ultraviolet (UV), ionizing radiation or different anticancer drugs, such as cisplatin, roscovitine and doxorubicin (DOX). Indeed, like p53, HIPK2 has been shown to contribute to the effectiveness of these treatments. Interestingly, p53-independent mechanisms of HIPK2-induced apoptosis were described for UV and tumor growth factor-β treatments; however, it is unknown whether these mechanisms are relevant for the responses to anticancer drugs. Because of the importance of the so-called 'p53-independent apoptosis and drug response' in human cancer chemotherapy, we asked whether p53-independent factor(s) might be involved in HIPK2-mediated chemosensitivity. Here, we show that HIPK2 depletion by RNA interference induces resistance to different anticancer drugs even in p53-null cells, suggesting the involvement of HIPK2 targets other than p53 in response to chemotherapy. In particular, we found that HIPK2 phosphorylates and promotes proteasomal degradation of ΔNp63α, a prosurvival ΔN isoform of the p53 family member, p63. Indeed, effective cell response to different genotoxic agents was shown to require phosphorylation-induced proteasomal degradation of ΔNp63α. In DOX-treated cells, we show that HIPK2 depletion interferes with ΔNp63α degradation, and expression of a HIPK2-resistant ΔNp63α-Δ390 mutant induces chemoresistance. We identify T397 as the ΔNp63α residue phosphorylated by HIPK2, and show that the non-phosphorylatable ΔNp63α-T397A mutant is not degraded in the face of either HIPK2 overexpression or DOX treatment. These results indicate ΔNp63α as a novel target of HIPK2 in response to genotoxic drugs.

Phosphorylation of TCF Proteins by Homeodomain-interacting Protein Kinase 2

Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos.

Ubiquitination and Degradation of Homeodomain-interacting Protein Kinase 2 by WD40 Repeat/SOCS Box Protein WSB-1

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses.

HIPK2 represses β-catenin-mediated transcription, epidermal stem cell expansion, and skin tumorigenesis

Transcriptional control by beta-catenin and lymphoid enhancer-binding factor 1 (LEF1)/T cell factor regulates proliferation in stem cells and tumorigenesis. Here we provide evidence that transcriptional co repressor homeodomain interacting protein kinase 2 (HIPK2) controls the number of stem and progenitor cells in the skin and the susceptibility to develop squamous cell carcinoma. Loss of HIPK2 leads to increased proliferative potential, more rapid G1-S transition in cell cycle, and expansion of the epidermal stem cell compartment. Among the critical regulators of G1-S transition in the cell cycle, only cyclin D1 is selectively up-regulated in cells lacking HIPK2. Conversely, overexpression of HIPK2 suppresses LEF1/beta-catenin-mediated transcriptional activation of cyclin D1 expression. However, deletion of the C-terminal YH domain of HIPK2 completely abolishes its ability to recruit another transcriptional corepressor CtBP and suppress LEF1/beta-catenin-mediated transcription. To determine whether loss of HIPK2 leads to increased susceptibility to tumorigenesis, we treat wild-type, Hipk2+/-, andHipk2-/- mice with the two-stage carcinogenesis protocol. Our results indicate that more skin tumors are induced in Hipk2+/- and Hipk2-/- mutants, with most of the tumors showing shortened incubation time and malignant progression. Together, our results indicate that HIPK2 is a tumor suppressor that controls proliferation by antagonizing LEF1/beta-catenin-mediated transcription. Loss of HIPK2 synergizes with activation of H-ras to induce tumorigenesis.

High-mobility group A1 inhibits p53 by cytoplasmic relocalization of its proapoptotic activator HIPK2

High-mobility group A1 (HMGA1) overexpression and gene rearrangement are frequent events in human cancer, but the molecular basis of HMGA1 oncogenic activity remains unclear. Here we describe a mechanism through which HMGA1 inhibits p53-mediated apoptosis by counteracting the p53 proapoptotic activator homeodomain-interacting protein kinase 2 (HIPK2). We found that HMGA1 overexpression promoted HIPK2 relocalization in the cytoplasm and inhibition of p53 apoptotic function, while HIPK2 overexpression reestablished HIPK2 nuclear localization and sensitivity to apoptosis. HIPK2 depletion by RNA interference suppressed the antiapoptotic effect of HMGA1, which indicates that HIPK2 is the target required for HMGA1 to repress the apoptotic activity of p53. Consistent with this process, a strong correlation among HMGA1 overexpression, HIPK2 cytoplasmic localization, and low spontaneous apoptosis index (comparable to that observed in mutant p53-carrying tumors) was observed in WT p53-expressing human breast carcinomas. Hence, cytoplasmic relocalization of HIPK2 induced by HMGA1 overexpression is a mechanism of inactivation of p53 apoptotic function that we believe to be novel.

Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta–gonad–mesonephros hematopoiesis

Definitive hematopoiesis starts in the aorta–gonad–mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45 + hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45 lowc-Kit + cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.