Homeodomain-interacting Protein Kinase Research Articles

Virtual Screening, Molecular Dynamics, and Mechanism Study of Homeodomain-Interacting Protein Kinase 2 Inhibitor in Renal Fibroblasts.

Background/Objectives: Homeodomain-interacting protein kinase 2 (HIPK2) is critically involved in the progression of renal fibrosis. This study aims to identify and characterize a novel HIPK2 inhibitor, CHR-6494, and investigate its therapeutic potential. Methods: Using structure-based virtual screening and molecular dynamics simulations, we identified CHR-6494 as a potent HIPK2 inhibitor with an IC50 of 0.97 μM. The effects of CHR-6494 on the phosphorylation of p53 in Normal Rattus norvegicus kidney cells (NRK-49F) induced by transforming growth factor-β (TGF-β) were assessed, along with its impact on TGF-β signaling and downstream profibrotic markers. Results: CHR-6494 significantly reduces p53 phosphorylation induced by TGF-β and enhances the interaction between HIPK2 and seven in absentia 2 (SIAH2), facilitating HIPK2 degradation via proteasomal pathways. Both CHR-6494 and Abemaciclib inhibit NRK-49F cell proliferation and migration induced by TGF-β, suppressing TGF-β/Smad3 signaling and decreasing profibrotic markers such as Fibronectin I (FN-I) Collagen I and α-smooth muscle actin (α-SMA). Additionally, these compounds inhibit nuclear factor kappa-B (NF-κB) signaling and reduce inflammatory cytokine expression. Conclusions: The study highlights the dual functionality of HIPK2 kinase inhibitors like CHR-6494 and Abemaciclib as promising therapeutic candidates for renal fibrosis and inflammation. The findings provide new insights into HIPK2 inhibition mechanisms and suggest pathways for the design of novel HIPK2 inhibitors in the future.

Genipin 1-O-β-D-gentiobioside ameliorates CUMS-induced prefrontal cortex neuron neuronal apoptosis by modulating HIPK2 SUMOylation

BackgroundDepression is a common mental illness with more than 280 million sufferers worldwide. Inflammation, particularly the c-Jun amino-terminal kinase (JNK) pathway, contributes to depression development and neuronal apoptosis. Gardenia is a herb with therapeutic effects on depression that has been shown to inhibit neuronal apoptosis. However, one of the components in gardenia, Genipin 1-O-β-D-gentiobioside(GG), has been less studied for its mechanism on depression. Thus, in the current study, we investigate how Genipin 1-O-β-D-gentiobioside improves depression and elucidate its possible mechanism of action. MethodsIn this investigation, we utilize a chronic unpredictable mild stress (CUMS) mouse model and corticosterone-induced primary cortical neurons to examine the role of GG in ameliorating depressive symptoms and neuronal apoptosis. TUNEL staining and flow cytometry assessed the effects of GG on neuronal apoptosis. Western Blot analyses and immunofluorescence assays apoptosis-related proteins in the prefrontal cortex and primary neurons. The site of action of GG in regulating homeodomain interacting protein kinase 2 (HIPK2) SUMOylation was further explored in primary neurons. We constructed siRNA-SUMO1 vectors to transfect primary neuronal cells with intracellular SUMO1 knockdown. Proximity ligation assay (PLA) experiments were performed on primary neurons according to the instructions of the assay kit to observe the physical relationship between HIPK2 and SUMO1. We predicted the HIPK2 SUMOylation modification site by an online database and constructed vectors to target and site-directed mutagenesis, then to transfected primary neuronal cells. ResultsThe results showed that GG effectively alleviated depressive-like behaviours, down-regulated apoptosis-related proteins (p-JNK, Bax, Cleaved-Caspase-3), and inhibited neuronal apoptosis in CUMS-induced depressed mice and corticosterone-induced primary cortical neurons. We reveal a complex mechanism underlying the link between GG, SUMOylation of HIPK2, and complex pathways of neuronal apoptosis regulation. K326 and K1189 are the key SUMOylation sites regulated by GG in intricate interactions of apoptosis-related proteins. ConclusionOur study demonstrated that GG exerts antidepressant-like actions through neuroprotective effects by inhibiting the apoptosis of prefrontal cortex neurons, revealing the mechanism of GG inhibition of JNK phosphorylation by enhancing HIPK2 SUMOylation.

Abstract We061: Cardiomyocytes HIPK2 Regulates Myocardial Inflammation And Heart Function Through Purinergic Signaling

Background: Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that belongs to an evolutionary conserved HIPK family of kinases. HIPK2-mediated regulation of the inflammation axis of signaling is a comparatively new concept. Purinergic signaling is critical to myocardial inflammation and function. However, there is no literature on HIPK2 regulating purinergic signaling. Methods and Results: Herein, we identified HIPK2, as a novel regulator of purinergic signaling. Cardiomyocyte-specific HIPK2 knockout (CM-HIPK2-KO) hearts exhibited reduced expression of multiple key players of canonical purinergic signaling, including ectonucleases CD39 and CD73, and led to excessive inflammation and cardiac dysfunction. Multiple in vitro experiments with gain-of-function (adenovirus expressing HIPK2, Ad-HIPK2), and loss-of-function (Ad-sh-RNA-HIPK2) approaches were performed to establish that HIPK2-mediated regulation of purinergic signaling is a conserved mechanism in many cell types of diverse backgrounds. Mechanistically, we identified that the signaling circuit of HIPK2-ERK-CREB exerts its effects on purinergic signaling through transcriptional control on CD39 and CD73. Aberrant activation of purinergic signaling and inflammation was critical to cardiac pathologies because interventions with purinergic signaling inhibitor apyrase or NLRP3 inflammasome inhibitor CY09 largely rescued the detrimental cardiac phenotype of CM-HIPK2 KOs. Conclusion: Herein, we identified HIPK2 as a novel regulator of purinergic signaling. Its deletion leads to excessive inflammation and cardiac dysfunction. Therefore, strategies to maintain HIPK2 are critical to sustaining cardiac health.

USP7-stabilised HIPK2 promotes high glucose-induced endothelial cell dysfunctions to accelerate diabetic foot ulcers

Background: This study aimed to explore the molecular mechanism of homeodomain-interacting protein kinase 2 (HIPK2) in diabetic foot ulcers (DFU). Methods: High glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were used to construct DFU cell models. Cell functions were determined using CCK8 assay, EdU assay, flow cytometry, transwell assay, wound healing assay and tube formation assay. Quantitative real-time PCR and western blot were applied to measure the gene expression. Results: HG treatment suppressed HUVECs proliferation, invasion, migration, and angiogenesis, while enhanced apoptosis. HIPK2 was overexpressed in DFU patients, and its knockdown alleviated HG-induced HUVECs dysfunctions. USP7 stabilised HIPK2 protein by reducing its ubiquitination. USP7 overexpression promoted HG-induced HUVECs dysfunctions, and HIPK2 upregulation also reversed the regulation of USP7 knockdown on HG-induced HUVECs dysfunctions. USP7/HIPK2 axis inhibited the activity of PI3K/AKT pathway. Conclusion: Our study revealed that USP7-stabilised HIPK2 contributed to HG-induced HUVECs dysfunctions, thus accelerating DFU process.

HIPK2 in Colon Cancer: A Potential Biomarker for Tumor Progression and Response to Therapies.

Colon cancer, one of the most common and fatal cancers worldwide, is characterized by stepwise accumulation of specific genetic alterations in tumor suppressor genes or oncogenes, leading to tumor growth and metastasis. HIPK2 (homeodomain-interacting protein kinase 2) is a serine/threonine protein kinase and a "bona fide" oncosuppressor protein. Its activation inhibits tumor growth mainly by promoting apoptosis, while its inactivation increases tumorigenicity and resistance to therapies of many different cancer types, including colon cancer. HIPK2 interacts with many molecular pathways by means of its kinase activity or transcriptional co-repressor function modulating cell growth and apoptosis, invasion, angiogenesis, inflammation and hypoxia. HIPK2 has been shown to participate in several molecular pathways involved in colon cancer including p53, Wnt/β-catenin and the newly identified nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2). HIPK2 also plays a role in tumor-host interaction in the tumor microenvironment (TME) by inducing angiogenesis and cancer-associated fibroblast (CAF) differentiation. The aim of this review is to assess the role of HIPK2 in colon cancer and the underlying molecular pathways for a better understanding of its involvement in colon cancer carcinogenesis and response to therapies, which will likely pave the way for novel colon cancer therapies.

Determination of homeodomain interacting protein kinase 2 polymorphisms rs2058265, rs6464214, and rs7456421 in patients with kidney stone

Determination of homeodomain interacting protein kinase 2 polymorphisms rs2058265, rs6464214, and rs7456421 in patients with kidney stone

Verbascoside inhibits oral squamous cell carcinoma cell proliferation, migration, and invasion by the methyltransferase 3-mediated microRNA-31-5p/homeodomain interacting protein kinase 2 axis

ObjectiveThe study aimed to investigate the effects of verbascoside on oral squamous cell carcinoma (OSCC) cellular behaviors and underlying molecular mechanisms. DesignFor this purpose, SCC9 and UM1 cell lines were treated with verbascoside, and their biological behaviors, including proliferation, migration, and invasion, were evaluated using cell counting kit-8, 5-Ethynyl-2′-deoxyuridine, and Transwell assays. The expression of methyltransferase-3 (METTL3), microRNA (miR)− 31–5p, and homeodomain interacting protein kinase-2 (HIPK2) were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between METTL3 and miR-31–5p was evaluated by RNA immunoprecipitation and methylated RNA immunoprecipitation, while the interaction between miR-31–5p and HIPK2 was evaluated by dual-luciferase reporter analysis. ResultsThe results indicated inhibition of OSCC cell proliferation, migration, and invasion post verbascoside treatment. Similarly, METTL3 was upregulated in OSCC cells and was inhibited post-verbascoside treatment. Overexpressing METTL3 promoted the cellular processes. Moreover, miR-31–5p was upregulated in OSCC cells, where METTL3 facilitated the processing of miR-31–5p in an N6-methyladenosine (m6A)-dependent manner. The HIPK2 served as miR-31–5p target, where overexpressing miR-31–5p or HIPK2 knockdown reversed the suppression of verbascoside-induced biological behaviors. ConclusionsVerbascoside inhibited the progression of OSCC by inhibiting the METTL3-regulated miR-31–5p/HIPK2 axis. These findings suggested that verbascoside might be an effective drug for OSCC therapy.

Upregulated lncRNA PRNT promotes progression and oxaliplatin resistance of colorectal cancer cells by regulating HIPK2 transcription.

Colorectal cancer (CRC) is the third most common cancer and a significant cause of cancer-related mortality globally. Resistance to chemotherapy, especially during CRC treatment, leads to reduced effectiveness of drugs and poor patient outcomes. Long noncoding RNAs (lncRNAs) have been implicated in various pathophysiological processes of tumor cells, including chemotherapy resistance, yet the roles of many lncRNAs in CRC remain unclear. To identify and analyze the lncRNAs involved in oxaliplatin resistance in CRC and to understand the underlying molecular mechanisms influencing this resistance. Gene Expression Omnibus datasets GSE42387 and GSE30011 were reanalyzed to identify lncRNAs and mRNAs associated with oxaliplatin resistance. Various bioinformatics tools were employed to elucidate molecular mechanisms. The expression levels of lncRNAs and mRNAs were assessed via quantitative reverse transcription-polymerase chain reaction. Functional assays, including MTT, wound healing, and Transwell, were conducted to investigate the functional implications of lncRNA alterations. Interactions between lncRNAs and transcription factors were examined using RIP and luciferase reporter assays, while Western blotting was used to confirm downstream pathways. Additionally, a xenograft mouse model was utilized to study the in vivo effects of lncRNAs on chemotherapy resistance. LncRNA prion protein testis specific (PRNT) was found to be upregulated in oxaliplatin-resistant CRC cell lines and negatively correlated with homeodomain interacting protein kinase 2 (HIPK2) expression. PRNT was demonstrated to sponge transcription factor zinc finger protein 184 (ZNF184), which in turn could regulate HIPK2 expression. Altered expression of PRNT influenced CRC cell sensitivity to oxaliplatin, with overexpression leading to decreased sensitivity and decreased expression reducing resistance. Both RIP and luciferase reporter assays indicated that ZNF184 and HIPK2 are targets of PRNT. The PRNT/ZNF184/HIPK2 axis was implicated in promoting CRC progression and oxaliplatin resistance both in vitro and in vivo. The study concludes that PRNT is upregulated in oxaliplatin-resistant CRC cells and modulates the expression of HIPK2 by sponging ZNF184. This regulatory mechanism enhances CRC progression and resistance to oxaliplatin, positioning PRNT as a promising therapeutic target for CRC patients undergoing oxaliplatin-based chemotherapy.

Tyr352 as a Predominant Phosphosite in the Understudied Kinase and Molecular Target, HIPK1: Implications for Cancer Therapy.

Homeodomain-interacting protein kinase 1 (HIPK1) is majorly found in the nucleoplasm. HIPK1 is associated with cell proliferation, tumor necrosis factor-mediated cellular apoptosis, transcription regulation, and DNA damage response, and thought to play significant roles in health and common diseases such as cancer. Despite this, HIPK1 remains an understudied molecular target. In the present study, based on a systematic screening and mapping approach, we assembled 424 qualitative and 44 quantitative phosphoproteome datasets with 15 phosphosites in HIPK1 reported across multiple studies. These HIPK1 phosphosites were not currently attributed to any functions. Among them, Tyr352 within the kinase domain was identified as the predominant phosphosite modulated in 22 differential datasets. To analyze the functional association of HIPK1 Tyr352, we first employed a stringent criterion to derive its positively and negatively correlated protein phosphosites. Subsequently, we categorized the correlated phosphosites in known interactors, known/predicted kinases, and substrates of HIPK1, for their prioritized validation. Bioinformatics analysis identified their significant association with biological processes such as the regulation of RNA splicing, DNA-templated transcription, and cellular metabolic processes. HIPK1 Tyr352 was also identified to be upregulated in Her2+ cell lines and a subset of pancreatic and cholangiocarcinoma tissues. These data and the systems biology approach undertaken in the present study serve as a platform to explore the functional role of other phosphosites in HIPK1, and by extension, inform cancer drug discovery and oncotherapy innovation. In all, this study highlights the comprehensive phosphosite map of HIPK1 kinase and the first of its kind phosphosite-centric analysis of HIPK1 kinase based on global-level phosphoproteomics datasets derived from human cellular differential experiments across distinct experimental conditions.

HIPK3 maintains sensitivity to platinum drugs and prevents disease progression in gastric cancer

In the realm of cancer therapeutics and resistance, kinases play a crucial role, particularly in gastric cancer (GC). Our study focused on platinum-based chemotherapy resistance in GC, revealing a significant reduction in homeodomain-interacting protein kinase 3 (HIPK3) expression in platinum-resistant tumors through meticulous analysis of transcriptome datasets. In vitro and in vivo experiments demonstrated that HIPK3 knockdown enhanced tumor proliferation and metastasis, while upregulation had the opposite effect. We identified the myocyte enhancer factor 2C (MEF2C) as a transcriptional regulator of HIPK3 and uncovered HIPK3's role in downregulating the morphogenesis regulator microtubule-associated protein (MAP7) through ubiquitination. Phosphoproteome profiling revealed HIPK3's inhibitory effects on mTOR and Wnt pathways crucial in cell proliferation and movement. A combined treatment strategy involving oxaliplatin, rapamycin, and IWR1-1-endo effectively overcame platinum resistance induced by reduced HIPK3 expression. Monitoring HIPK3 levels could serve as a GC malignancy and platinum resistance indicator, with our proposed treatment strategy offering novel avenues for reversing resistance in gastric cancer.

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.

Circ_0008410 contributes to fibroblast-like synoviocytes dysfunction by regulating miR-149-5p/HIPK2 axis.

Circular RNAs (circRNAs) play functional roles in rheumatoid arthritis (RA) progression. Fibroblast-like synoviocytes (RASFs) are the main effectors in RA development. In this study, we explored the function and mechanism of circ_0008410 in RASFs. qRT-PCR was used to detect the expression of circ_0008410, microRNA-149-5p (miR-149-5p), and homeodomain-interacting protein kinase 2 (HIPK2). Cell counting kit-8, EdU assay, flow cytometry, and transwell assay were performed to evaluate cell proliferation, apoptosis, migration, and invasion. Western blot measured the protein levels of related markers and HIPK2. The levels of IL-1β, TNF-α, and IL-6 were tested by corresponding ELISA kits and Western blot. The combination between miR-149-5p and circ_0008410 or HIPK2 was detected by dual-luciferase reporter assay or RNA immunoprecipitation (RIP) assay. Our data showed that circ_0008410 and HIPK2 were elevated, while miR-149-5p was downregulated in RA synovial tissues and RASFs. Circ_0008410 promoted RASF proliferation, migration, invasion, and inflammation while inhibiting apoptosis. MiR-149-5p was a target of circ_0008410, and its overexpression could reverse the promoting effects of circ_0008410 on RASF dysfunction. Moreover, miR-149-5p could target HIPK2 to suppress RASF proliferation, migration, invasion, and inflammation. Collectively, circ_0008410 promoted RASF dysfunction via miR-149-5p/HIPK2, which might provide a potential target for RA therapy.

Overexpression of microRNA-205-5p promotes cholangiocarcinoma growth by reducing expression of homeodomain-interacting protein kinase 3

The microRNA miR-205-5p has diverse effects in different malignancies, including cholangiocarcinoma (CCA), but its effects on CCA progression is unclear. Here we investigated the role and function of miR-205-5p in CCA. Three CCA cell lines and human serum samples were found to have much higher expression levels of miR-205-5p than seen in typical cholangiocyte cell lines and healthy controls. Inhibition of miR-205-5p suppressed CCA cell motility, invasion and proliferation of KKU-213B whereby overexpression of miR-205-5p promoted cell proliferation and motility of KKU-100 cells. Bioinformatics tools (miRDB, TargetScan, miRWalk, and GEPIA) all predicted various miR-205-5p targets. Experiments using miR-205-5p inhibitor and mimic indicated that homeodomain-interacting protein kinase 3 (HIPK3) was a potential direct target of miR-205-5p. Overexpression of HIPK3 using HIPK3 plasmid cloning DNA suppressed migration and proliferation of KKU-100 cells. Notably, HIPK3 expression was lower in human CCA tissues than in normal adjacent tissues. High HIPK3 expression was significantly associated with longer survival time of CCA patients. Multivariate regression analysis indicated tissue HIPK3 levels as an independent prognostic factor for CCA patients. These findings indicate that overexpression of miR-205-5p promotes CCA cells proliferation and migration partly via HIPK3-dependent way. Therefore, targeting miR-205-5p may be a potential treatment approach for CCA.

REST-restrained lncRNA EPB41L4A-AS2 Modulates Laryngeal Squamous Cell Carcinoma Development via Regulating miR-1254/HIPK2 Pathway.

LncRNAs have been corroborated to exert crucial effects in malignancies, including laryngeal squamous cell carcinoma (LSCC). Nevertheless, the role and mechanism of EPB41L4A- AS2 in LSCC are inadequately investigated and warrant further exploration. Relevant database was adopted to analyze the relationship between EPB41L4A-AS2 expression level and tumors. The expressions and relationships of EPB41L4A-AS2, RE-1 silencing transcription factor (REST), miR-1254, and homeodomain interacting protein kinase 2 (HIPK2) in LSCC cells were evaluated by qRT-PCR, Pearson's correlation tests, RNA immunoprecipitation, RNA pull-down assay, chromatin immunoprecipitation, database, and dual-luciferase reporter assay. Following the required transfection, the biological behaviors of LSCC cells were examined using cell function experiments. Meanwhile, the levels of Ki-67 and apoptosis-, and epithelial-mesenchymal transition (EMT) pathway-related proteins were quantified with Western blot. Moreover, xenografts in nude mice were constructed, and the tumor volume and weight were measured. Ki-67 positivity was determined by immunohistochemical staining. EPB41L4A-AS2 and HIPK2 were lower-expressed, yet miR-1254 and REST were higher- expressed in LSCC cells. Pearson's correlation assay results exhibited a positive correlation between HIPK2 and EPB41L4A-AS2 and a negative correlation between HIPK2 and miR-1254. Overexpressed EPB41L4A-AS2 diminished the biological behavior, and repressed the levels of Ki-67 and EMT-related markers in LSCC cells whilst enhancing those of apoptosis-related markers. These aforementioned effects were counteracted by miR-1254 mimic. Moreover, EPB41L4A- AS2 overexpression suppressed the growth of tumors and reduced the positive expression of Ki-67 in nude mice. Besides, miR-1254 aggravated the biological behaviors and elevated the levels of Ki-67 and EMT-related proteins in LSCC cells while reducing the levels of apoptosis-related markers via targeting HIPK2. REST-restrained EPB41L4A-AS2 modulates LSCC development via regulating miR-1254/HIPK2 pathway.

THU045 WIP1 Phosphatase Is A Novel Specific Target For Epithelial GH Action

Abstract Disclosure: T. Apaydin: None. S. Zonis: None. C. Zhou: None. R. Barrett: None. V. Chesnokova: None. S. Melmed: None. Deficient DNA repair and accumulated DNA damage underlie age-associated pathological changes, tissue fragility, and neoplastic development. ATM kinase, an activator of DNA damage responses (DDR), phosphorylates DDR effectors including CHK2, p53, and γH2AX to ensure DNA repair. We have shown that GH suppresses epithelial DDR activity by suppressing ATM phosphorylation, resulting in DNA damage accumulation due to repressed DNA repair. Wip1 dephosphorylates ATM thereby attenuating DNA damage repair. We found that GH significantly induced Wip1 expression (1.6-fold) in both human normal colon cells (hNCC) and in human 3-dimensional intestinal organoids, resulting in dephosphorylated ATM, CHK2, γH2AX, and p53. Mice bearing GH-secreting tumors with high circulating GH also exhibited induced colon Wip1 expression with lower phospho-ATM, γH2AX, and p53 as well as increased DNA damage. Twofold higher Wip1 expression was observed in blood buffy coats derived from patients with GH-secreting adenoma vs patients with non-functioning pituitary adenoma. Specificity of these observations was validated by showing that in GH-treated cells, blocking GH receptor (GHR) with pegvisomant or blocking STAT5 phosphorylation with the inhibitor CAS 285986-31-4 constrained Wip1 induction and restored ATM phosphorylation, indicating that GH effects on Wip1 are GHR/STAT5-mediated concordant with the observed decreased colon Wip1 expression in GHR-/- mice devoid of GH signaling. Furthermore, Wip1 inhibition with the small molecule inhibitor (GSK2830371) reversed GH-induced DNA damage by restoring phosphorylation of ATM and other key DDR proteins. To determine a mechanism for Wip1 induction, we tested homeodomain-interacting protein kinase 2 (HIPK2) which is regulated by AMPK and potentiates Wip1 proteasomal degradation and its activity. We observed that GH increased phospho-AMPK expression, while blocking AMPK kinase activity with Compound C abrogated GH induction of Wip1. Src upregulates AMPK and triggers HIPK2 cytoplasmic relocation thereby inactivating HIPK2. We found that GH, by activating Src/AMPK phosphorylation, elicits HIPK2 cytoplasmic relocation, thus suppressing Wip1 ubiquitination and increasing its stability. Consistent with these findings, blocking Src phosphorylation with the tyrosine kinase inhibitor dasatinib abolished Wip1 upregulation and induced phosphorylation of its downstream targets. These results indicate that GH suppresses DDR by inducing Wip1 expression, and suppression of Wip1 may prevent epithelial accumulation of unrepaired DNA damage. These findings identify Wip1 as a specific novel target for GH action mediating GH-attenuation of DNA repair. Presentation: Thursday, June 15, 2023

The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model.

Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. We find that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, our research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth.

ZC3H13 Accelerates Keloid Formation by Mediating N6-methyladenosine Modification of HIPK2.

Keloids are fibroproliferative skin disorders caused by the improper healing of wounded skin. A growing body of evidence suggests the involvement of N6-Methyladenosine (m6A) modification in various bioprocesses; however, its role in keloid formation has not yet been investigated. The aim of this study was to determine the effect of the m6A regulator zinc finger CCCH domain containing protein 13 (ZC3H13) on the pathogenesis of keloid formation. ZC3H13 and homeodomain-interacting protein kinase 2 (HIPK2) expression was evaluated in healthy skin and keloid tissues, as well as in human dermal fibroblasts and human keloid fibroblasts (HKF), using qRT-PCR and western blotting. The effects of ZC3H13 overexpression and knockdown on the cell function of HKFs were assessed using CCK8, transwell, and flow cytometry. Furthermore, the influence of ZC3H13 on HIPK2 m6A modification was assessed using MeRIP-qPCR and mRNA stability assays. Both ZC3H13 expression and m6A RNA methylation were upregulated in keloid tissues and HKFs. Silencing of ZC3H13 inhibited proliferation and migration, while enhancing apoptosis in HKFs, whereas overexpression had the opposite effect. Furthermore, HIPK2 levels were high in keloid tissues and HKFs, and a positive correlation was observed between ZC3H13 and HIPK2. In HKFs, ZC3H13 overexpression elevated the m6A levels of HIPK2 mRNA and reduced the rate of HIPK2 mRNA degradation. Mechanically, ZC3H13-induced m6A modifications significantly improved HIPK2 mRNA stability. Collectively, ZC3H13 accelerated keloid formation by mediating the m6A modification of HIPK2 mRNA and maintaining its stability.

Adipose-derived stem cell exosome NFIC improves diabetic foot ulcers by regulating miR-204-3p/HIPK2

BackgroundDiabetic foot ulcers (DFU) are a serious complication of diabetes that lead to significant morbidity and mortality. Recent studies reported that exosomes secreted by human adipose tissue-derived mesenchymal stem cells (ADSCs) might alleviate DFU development. However, the molecular mechanism of ADSCs-derived exosomes in DFU is far from being addressed.MethodsHuman umbilical vein endothelial cells (HUVECs) were induced by high-glucose (HG), which were treated with exosomes derived from nuclear factor I/C (NFIC)-modified ADSCs. MicroRNA-204-3p (miR-204-3p), homeodomain-interacting protein kinase 2 (HIPK2), and NFIC were determined using real-time quantitative polymerase chain reaction. Cell proliferation, apoptosis, migration, and angiogenesis were assessed using cell counting kit-8, 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, wound healing, and tube formation assays. Binding between miR-204-3p and NFIC or HIPK2 was predicted using bioinformatics tools and validated using a dual-luciferase reporter assay. HIPK2, NFIC, CD81, and CD63 protein levels were measured using western blot. Exosomes were identified by a transmission electron microscope and nanoparticle tracking analysis.ResultsmiR-204-3p and NFIC were reduced, and HIPK2 was enhanced in DFU patients and HG-treated HUVECs. miR-204-3p overexpression might abolish HG-mediated HUVEC proliferation, apoptosis, migration, and angiogenesis in vitro. Furthermore, HIPK2 acted as a target of miR-204-3p. Meanwhile, NFIC was an upstream transcription factor that might bind to the miR-204-3p promoter and improve its expression. NFIC-exosome from ADSCs might regulate HG-triggered HUVEC injury through miR-204-3p-dependent inhibition of HIPK2.ConclusionExosomal NFIC silencing-loaded ADSC sheet modulates miR-204-3p/HIPK2 axis to suppress HG-induced HUVEC proliferation, migration, and angiogenesis, providing a stem cell-based treatment strategy for DFU.

MiR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression.

MicroRNA-21 (miR-21) plays an anti-apoptotic role following ischemia-reperfusion (I/R) injury (IRI) in vivo; however, its underlying mechanism remains unclear. This study explored the effects of miR-21 and homeodomain interacting protein kinase 3 (HIPK3) on cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) in vitro. To this end, the rat cardiomyocyte H9C2 cell line was exposed to H/R and the roles of miR-21 and HIPK3 in regulating cell viability and apoptosis were evaluated by cell counting kit-8 assay, terminal-deoxynucleotidyl-transferase -mediated dUTP nick end labeling, and flow cytometry. Immunofluorescence and western blotting were performed to detect the expression/phosphorylation of apoptosis-related proteins. miR-21 expression was measured with quantitative real-time polymerase chain reaction. The putative interaction between miR-21 and HIPK3 was evaluated using the luciferase reporter assay. Our results showed that: (i) miR-21 overexpression or HIPK3 downregulation significantly attenuated H9C2 cells apoptosis after H/R; (ii) suppression of miR-21 expression promoted apoptosis; (iii) miR-21 overexpression inhibited HIPK3 expression; (iv) HIPK3 was the direct and main target of miR-21; (v) miR-21/HIPK3 formed part of a reciprocal, negative feedback loop; and (vi) HIPK3 downregulation decreased FAS-mediated apoptosis by inhibiting the phosphorylation of FADD, which subsequently inhibited the expression of BAX and cleaved caspase-3 and increased the expression of BCL2. Our study indicates that miR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression, which could eventually have important clinical implications for patients with acute myocardial infarction.

Development of a circHIPK3-based ceRNA network and identification of mRNA signature in breast cancer patients harboring BRCA mutation.

Exploring the regulatory network of competing endogenous RNAs (ceRNAs) as hallmarks for breast cancer development has great significance and could provide therapeutic targets. An mRNA signature predictive of prognosis and therapy response in BRCA carriers was developed according to circular RNA homeodomain-interacting protein kinase 3 (circHIPK3)-based ceRNA network. We constructed a circHIPK3-based ceRNA network based on GSE173766 dataset and identified potential mRNAs that were associated with BRCA mutation patients within this ceRNA network. A total of 11 prognostic mRNAs and a risk model were identified and developed by univariate Cox regression analysis and the LASSO regression analysis as well as stepAIC method. Genomic landscape was treated by mutect2 and fisher. Immune characteristics was analyzed by ESTIMATE, MCP-counter. TIDE analysis was conducted to predict immunotherapy. The clinical treatment outcomes of BRCA mutation patients were assessed using a nomogram. The proliferation, migration and invasion in breast cancer cell lines were examined using CCK8 assay and transwell assay. We found 241 mRNAs within the circHIPK3-based ceRNA network. An 11 mRNA-based signature was identified for prognostic model construction. High risk patients exhibited dismal prognosis, low response to immunotherapy, less immune cell infiltration and tumor mutation burden (TMB). High-risk patients were sensitive to six anti-tumor drugs, while low-risk patient were sensitive to 47 drugs. The risk score was the most effective on evaluating patients' survival. The robustness and good prediction performance were validated in The Cancer Genome Atlas (TCGA) dataset and immunotherapy datasets, respectively. In addition, circHIPK3 mRNA level was upregulated, and promoted cell viability, migration and invasion in breast cancer cell lines. The current study could improve the understanding of mRNAs in relation to BRCA mutation and pave the way to develop mRNA-based therapeutic targets for breast cancer patients with BRCA mutation.