Paxillin family proteins Hic-5 and LPXN promote lipid storage by regulating the ubiquitination degradation of CIDEC.

Many metabolic diseases are caused by disorders of lipid homeostasis. CIDEC, a lipid droplet (LD)-associated protein, plays a critical role in controlling LD fusion and lipid storage. However, regulators of CIDEC remain largely unknown. Here, we established a homogeneous time-resolved fluorescence (HTRF)-based high-throughput screening method and identified LPXN as a positive regulatory candidate for CIDEC. LPXN and Hic-5, the members of the Paxillin family, are focal adhesion adaptor proteins that contribute to the recruitment of specific kinases and phosphatases, cofactors, and structural proteins, participating in the transduction of extracellular signals into intracellular responses. Our data showed that Hic-5 and LPXN significantly increased the protein level of CIDEC and enhanced CIDEC stability not through triacylglycerol synthesis and FAK signaling pathways. Hic-5 and LPXN reduced the ubiquitination of CIDEC and inhibited its proteasome degradation pathway. Furthermore, Hic-5 and LPXN enlarged LDs and promoted lipid storage in adipocytes. Therefore, we identified Hic-5 and LPXN as novel regulators of CIDEC. Our current findings also suggest intervention with Hic-5 and LPXN might ameliorate ectopic fat storage by enhancing the lipid storage capacity of white adipose tissues.

Hic-5 deficiency protects cerulein-induced chronic pancreatitis via down-regulation of the NF-κB (p65)/IL-6 signalling pathway.

Chronic pancreatitis (CP), characterized by pancreatic fibrosis, is a recurrent, progressive and irreversible disease. Activation of the pancreatic stellate cells (PSCs) is considered a core event in pancreatic fibrosis. In this study, we investigated the role of hydrogen peroxide-inducible clone-5 (Hic-5) in CP. Analysis of the human pancreatic tissue samples revealed that Hic-5 was overexpressed in patients with CP and was extremely low in healthy pancreas. Hic-5 was significant up-regulated in the activated primary PSCs independently from transforming growth factor beta stimulation. CP induced by cerulein injection was ameliorated in Hic-5 knockout (KO) mice, as shown by staining of tissue level. Simultaneously, the activation ability of the primary PSCs from Hic-5 KO mice was significantly attenuated. We also found that the Hic-5 up-regulation by cerulein activated the NF-κB (p65)/IL-6 signalling pathway and regulated the downstream extracellular matrix (ECM) genes such as α-SMA and Col1a1. Therefore, we determined whether suppressing NF-κB/p65 alleviated CP by treating mice with the NF-κB/p65 inhibitor triptolide in the cerulein-induced CP model and found that pancreatic fibrosis was alleviated by NF-κB/p65 inhibition. These findings provide evidence for Hic-5 as a therapeutic target that plays a crucial role in regulating PSCs activation and pancreatic fibrosis.

Increased expression of the transforming growth factor ß-inducible gene HIC-5 in systemic sclerosis skin and fibroblasts: a novel antifibrotic therapeutic target.

OBJECTIVE: SSc is a systemic fibrotic disease affecting skin, numerous internal organs and the microvasculature. The molecular pathogenesis of SSc tissue fibrosis has not been fully elucidated, although TGF-ß1 plays a crucial role. The Hic-5 protein encoded by the TGF-ß1-inducible HIC-5 gene participates in numerous TGF-ß-mediated pathways, however, the role of Hic-5 in SSc fibrosis has not been investigated. The aim of this study was to examine HIC-5 involvement in SSc tissue fibrosis. METHODS: Affected skin from three patients with diffuse SSc and dermal fibroblasts cultured from affected and non-affected SSc skin were examined for HIC-5 and COL1A1 gene expression. Real-time PCR, IF microscopy, western blotting and small interfering RNA-mediated HIC-5 were performed. RESULTS: HIC-5 and COL1A1 transcripts and Hic-5, type 1 collagen (COL1) and α-smooth muscle actin (α-SMA) protein levels were increased in clinically affected SSc skin compared with normal skin and in cultured dermal fibroblasts from affected SSc skin compared with non-affected skin fibroblasts from the same patients. HIC-5 knockdown caused a marked reduction of COL1 production in SSc dermal fibroblasts. CONCLUSION: HIC-5 expression is increased in affected SSc skin compared with skin from normal individuals. Affected SSc skin fibroblasts display increased HIC-5 and COL1A1 expression compared with non-affected skin fibroblasts from the same patients. Hic-5 protein was significantly increased in cultured SSc dermal fibroblasts. HIC-5 mRNA knockdown in SSc fibroblasts caused >50% reduction of COL1 production. Although these are preliminary results owing to the small number of skin samples studied, they indicate that Hic-5 plays a role in the profibrotic activation of SSc dermal fibroblasts and may represent a novel molecular target for antifibrotic therapy in SSc.

Glucocorticoid receptor binding to chromatin is selectively controlled by the coregulator Hic-5 and chromatin remodeling enzymes.

The steroid hormone-activated glucocorticoid receptor (GR) regulates cellular stress pathways by binding to genomic regulatory elements of target genes and recruiting coregulator proteins to remodel chromatin and regulate transcription complex assembly. The coregulator hydrogen peroxide-inducible clone 5 (Hic-5) is required for glucocorticoid (GC) regulation of some genes but not others and blocks the regulation of a third gene set by inhibiting GR binding. How Hic-5 exerts these gene-specific effects and specifically how it blocks GR binding to some genes but not others is unclear. Here we show that site-specific blocking of GR binding is due to gene-specific requirements for ATP-dependent chromatin remodeling enzymes. By depletion of 11 different chromatin remodelers, we found that ATPases chromodomain helicase DNA-binding protein 9 (CHD9) and Brahma homologue (BRM, a product of the SMARCA2 gene) are required for GC-regulated expression of the blocked genes but not for other GC-regulated genes. Furthermore, CHD9 and BRM were required for GR occupancy and chromatin remodeling at GR-binding regions associated with blocked genes but not at GR-binding regions associated with other GC-regulated genes. Hic-5 selectively inhibits GR interaction with CHD9 and BRM, thereby blocking chromatin remodeling and robust GR binding at GR-binding sites associated with blocked genes. Thus, Hic-5 regulates GR binding site selection by a novel mechanism, exploiting gene-specific requirements for chromatin remodeling enzymes to selectively influence DNA occupancy and gene regulation by a transcription factor.

Hic-5 is required for myofibroblast differentiation by regulating mechanically dependent MRTF-A nuclear accumulation.

How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-ß on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-ß. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-ß. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-ß.

Hic-5 deficiency attenuates the activation of hepatic stellate cells and liver fibrosis through upregulation of Smad7 in mice.

BACKGROUND & AIM: Hydrogen peroxide-inducible clone-5 (Hic-5), also named as transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was found to be induced by TGF-ß. Previous studies have shown that TGF-ß is a principal mediator of hepatic stellate cell (HSC) activation in liver fibrosis. However, this process remains elusive. In this study, we aimed to define the role of Hic-5 in HSC activation and liver fibrosis. METHODS: We examined the expression levels of Hic-5 during HSCs activation and in fibrotic liver tissues by quantitative real-time reverse transcriptase polymerase chain reaction, Western blot and immunohistochemistry. Hic-5 knockout (KO) and wild-type (WT) mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) injection to induce liver fibrosis. RESULTS: Hic-5 expression was strongly upregulated in activated HSCs of the human fibrotic liver tissue and BDL or CCl4-induced mouse liver fibrosis. Hic-5 deficiency significantly attenuated mouse liver fibrosis and HSC activation. Furthermore, Hic-5 knockdown by siRNA in vivo repressed CCl4-induced liver fibrosis in mice. Mechanistically, the absence of Hic-5 significantly inhibited the TGF-ß/Smad2 signaling pathway, proved by increasing Smad7 expression, resulting in reduced collagen production and α-smooth muscle actin expression in the activated HSCs. CONCLUSION: Hic-5 deficiency attenuates the activation of HSCs and liver fibrosis though reducing the TGF-ß/Smad2 signaling by upregulation of Smad7. Thus, Hic-5 can be regarded as a potential therapeutic target for liver fibrosis.

Hic-5 Mediates TGFß-Induced Adhesion in Vascular Smooth Muscle Cells by a Nox4-Dependent Mechanism.

OBJECTIVE: Focal adhesions (FAs) link the cytoskeleton to the extracellular matrix and as such play important roles in growth, migration, and contractile properties of vascular smooth muscle cells. Recently, it has been shown that downregulation of Nox4, a transforming growth factor (TGF) ß-inducible, hydrogen peroxide (H2O2)-producing enzyme, affects the number of FAs. However, the effectors downstream of Nox4 that mediate FA regulation are unknown. The FA resident protein H2O2-inducible clone (Hic)-5 is H2O2 and TGFß inducible, and a binding partner of the heat shock protein (Hsp) 27. The objective of this study was to elucidate the mechanism, by which Hic-5 and Hsp27 participate in TGFß-induced, Nox4-mediated vascular smooth muscle cell adhesion and migration. APPROACH AND RESULTS: Through a combination of molecular biology and biochemistry techniques, we found that TGFß, by a Nox4-dependent mechanism, induces the expression and interaction of Hic-5 and Hsp27, which is essential for Hic-5 localization to FAs. Importantly, we found that Hic-5 expression is required for the TGFß-mediated increase in FA number, adhesive forces and migration. Mechanistically, Nox4 downregulation impedes Smad (small body size and mothers against decapentaplegic) signaling by TGFß, and Hsp27 and Hic-5 upregulation by TGFß is blocked in small body size and mothers against decapentaplegic 4-deficient cells. CONCLUSIONS: Hic-5 and Hsp27 are effectors of Nox4 required for TGFß-stimulated FA formation, adhesion strength and migration in vascular smooth muscle cell.

Negative regulation of NADPH oxidase 4 by hydrogen peroxide-inducible clone 5 (Hic-5) protein.

Hydrogen peroxide-inducible clone 5 (Hic-5) is a focal adhesion adaptor protein induced by the profibrotic cytokine TGF-ß1. We have demonstrated previously that TGF-ß1 induces myofibroblast differentiation and lung fibrosis by activation of the reactive oxygen species-generating enzyme NADPH oxidase 4 (Nox4). Here we investigated a potential role for Hic-5 in regulating Nox4, myofibroblast differentiation, and senescence. In normal human diploid fibroblasts, TGF-ß1 induces Hic-5 expression in a delayed manner relative to the induction of Nox4 and myofibroblast differentiation. Hic-5 silencing induced constitutive Nox4 expression and enhanced TGF-ß1-inducible Nox4 levels. The induction of constitutive Nox4 protein in Hic-5-silenced cells was independent of transcription and translation and controlled by the ubiquitin-proteasomal system. Hic-5 associates with the ubiquitin ligase Cbl-c and the ubiquitin-binding protein heat shock protein 27 (HSP27). The interaction of these proteins is required for the ubiquitination of Nox4 and for maintaining low basal levels of this reactive oxygen species-generating enzyme. Our model suggests that TGF-ß1-induced Hic-5 functions as a negative feedback mechanism to limit myofibroblast differentiation and senescence by promoting the ubiquitin-proteasomal system-mediated degradation of Nox4. Together, these studies indicate that endogenous Hic-5 suppresses senescence and profibrotic activities of myofibroblasts by down-regulating Nox4 protein expression. Additionally, these are the first studies, to our knowledge, to demonstrate posttranslational regulation of Nox4.

Preclinical trial of targeting the Hic-5-mediated pathway to prevent the progression of hepatocellular carcinoma.

The poor prognosis of hepatocellular carcinoma (HCC) was ascribed to metastasis. Targeted therapy aiming at the molecules along the metastatic pathway is a promising therapeutic strategy. Among them, hydrogen peroxide inducible clone-5 (Hic-5) is highlighted. Hic-5, discovered as a reactive oxygen species (ROS)-inducible gene, was identified to be an adaptor protein in focal adhesion and a critical signaling mediator upregulated in various cancers including HCC. Moreover, Hic-5 may regulate epithelial-mesenchymal transition (EMT) transcription factor Snail and its downstream mesenchymal genes including fibronectin and matrix metalloproteinase-9 required for migration and invasion of HCC. However, the comprehensive Hic-5-mediated pathway was not established and whether Hic-5 can be a target for preventing HCC progression has not been validated in vivo. Using whole-transcriptome mRNA sequencing, we found reactive oxygen species modulator (ROMO) and ZNF395 were upregulated by Hic-5 in a patient-derived HCC cell line, HCC372. Whereas ROMO was involved in Hic-5-mediated ROS signaling, ZNF395 locates downstream of Snail for mesenchymal genes expression required for cell migration. Also, ZNF395 but not ROMO was upregulated by Hic-5 for migration in another patient-derived HCC cell line, HCC374. Further, by in vivo knock down of Hic-5 using the Stable Nucleic Acids Lipid nanoparticles (SNALP)-carried Hic-5 siRNA, progression of HCC372 and HCC374 in SCID mice was prevented, coupled with the decrease of the downstream mesenchymal genes. Our study provides the preclinical evidence that targeting Hic-5 is potentially able to prevent the progression of HCCs with Hic-5 overexpression.

Jagged-1 is induced by mTOR inhibitors in renal cancer cells through an Akt/ALK5/Smad4-dependent mechanism.

The mammalian target of rapamycin (mTOR) plays an important role in the aggressiveness and therapeutic resistance of many cancers. Targeting mTOR continues to be under clinical investigation for cancer therapy. Despite the notable clinical success of mTOR inhibitors in extending the overall survival of patients with certain malignancies including metastatic renal cell carcinomas (RCCs), the overall impact of mTOR inhibitors on cancers has been generally disappointing and attributed to various compensatory responses. Here we provide the first report that expression of the Notch ligand Jagged-1 (JAG1), which is associated with aggressiveness of RCCs, is induced by several inhibitors of mTOR (rapamycin (Rap), BEZ235, KU-0063794) in human clear cell RCC (ccRCC) cells. Using both molecular and chemical inhibitors of PI3K, Akt, and TGF-ß signaling, we provide evidence that the induction of JAG1 expression by mTOR inhibitors in ccRCC cells depends on the activation of Akt and occurs through an ALK5 kinase/Smad4-dependent mechanism. Furthermore, we show that mTOR inhibitors activate Notch1 and induce the expression of drivers of epithelial-mesenchymal transition, notably Hic-5 and Slug. Silencing JAG1 with selective shRNAs blocked the ability of KU-0063794 and Rap to induce Hic-5 in ccRCC cells. Moreover, Rap enhanced TGF-ß-induced expression of Hic-5 and Slug, both of which were repressed in JAG1-silenced ccRCC cells. Silencing JAG1 selectively decreased the motility of ccRCC cells treated with Rap or TGF-ß1. Moreover, inhibition of Notch signaling with γ-secretase inhibitors enhanced or permitted mTOR inhibitors to suppress the motility of ccRCC cells. We suggest targeting JAG1 may enhance therapeutic responses to mTOR inhibitors in ccRCCs.

Hic-5 deficiency attenuates hepatic ischemia reperfusion injury through TLR4/NF-κB signaling pathways.

AIM: To explore the role and mechanism of Hydrogen peroxide-inducible clone-5 (Hic-5) in hepatic ischemia reperfusion injury. METHODS: Hic-5 KO and WT mice were used to establish the liver ischemia reperfusion model (HI/R). Primary hepatocytes were isolated to establish hypoxic reoxygenation model (H/R). AST and ALT were measured by automatic biochemical analyzer. Liver tissue sections were stained with HE and Tunnel. RNA and proteins were extracted from liver tissues, and expressions of Il-6, Il-10, CCL-2, CXCL-10, P65, Caspase-3, TLR4 and FADD were detected at gene and protein levels. Liver cell apoptosis was detected by flow cytometry and immunofluorescence. Primary hepatocytes were stimulated by LPS to establish a model of hepatocyte apoptosis, and cell inflammation and apoptosis-related proteins were detected. RESULTS: After HI/R, ALT and AST in serum were up-regulated, some hepatocyte apoptosis were observed in pathological sections. Hic-5 expression was increased in WT mice after HI/R, and liver damage were severer than KO mice. The expression of IL-6, CCL-2 and CXCL-10 in the liver of KO mice was low, and the expression of IL-10 was high. Further studies showed that KO mice showed lower expression of P65, Caspase3 and TLR4. In H/R model, hepatocytes also showed the same trend. Finally, after LPS stimulation, the results showed that the inflammation and apoptosis induced by LPS were significantly reduced in Hic-5 knocked hepatocytes. CONCLUSION: Hic-5 was found to promote inflammation through NF-kb signaling pathway and apoptosis through TLR4-FADD signaling pathway in mice with HI/R, thus aggravating liver injury in mice.

Exosome-mediated Hic-5 regulates proliferation and apoptosis of osteosarcoma via Wnt/ß-catenin signal pathway.

The expression of Hic-5 was detected in osteosarcoma patients and osteosarcoma cell lines by RT-PCR. Then RFP-sh-Hic-5 was transfected into osteosarcoma cell lines. The effect of Hic-5 on cell viability, proliferation and apoptosis were assessed by MTT, EdU kit and Flow cytometry. The exosomes were isolated from MG-63 cell supernatant by an Exosome Isolation Kit. The exosome-Hic-5 was confirmed by transmission electron microscope, particle size detection and RT-PCR. Next, exosome-Hic-5 treated cells were explored the cell viability, proliferation and apoptosis. Further, Co-IP assay was employed for identifying the relationship between Hic-5 and smad4. TCF/LEF and the protein level of components of wnt/ß-catenin signals were detected by TOP luciferase assay and western blot. Hic-5 was upregulated in osteosarcoma tissues and cell. Forced decreased expression Hic-5 inhibited the proliferation of osteosarcoma cell lines, and induced apoptosis of MG-63 and HOS. In vivo, silencing Hic-5 remitted the tumor progression. Further, we isolated the exosomes from MG-63 supernatant, exosomes concluding Hic-5 would regulated the proliferation and apoptosis level of MG-63 and HOS cells. Further, Hic-5 interacted with smad4 and regulated Wnt/ß-catenin signal by decreasing TCF/LEF activity. Silencing Hic-5 inhibited the proliferation and induced apoptosis of osteosarcoma cell via inactivating Wnt/ß-catenin signal by exosome pathway.

Paxillin family of focal adhesion adaptor proteins and regulation of cancer cell invasion.

The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are important in cell adhesion and migration of both normal and cancer cells. Historically, the role of these proteins in regulating the actin cytoskeleton through focal adhesion-mediated signaling has been well documented. However, studies in recent years have revealed additional functions in modulating the microtubule and intermediate filament cytoskeletons to affect diverse processes including cell polarization, vesicle trafficking and mechanosignaling. Expression of paxillin family proteins in stromal cells is also important in regulating tumor cell migration and invasion through non-cell autonomous effects on the extracellular matrix. Both paxillin and Hic-5 can also influence gene expression through a variety of mechanisms, while their own expression is frequently dysregulated in various cancers. Accordingly, these proteins may serve as valuable targets for novel diagnostic and treatment approaches in cancer.

Hic-5 in pancreatic stellate cells affects proliferation, apoptosis, migration, invasion of pancreatic cancer cells and postoperative survival time of pancreatic cancer.

Pancreatic cancer is one of the most severe types of tumors, with a 5-year survival rate of less than 7%. The prognosis and treatment of pancreatic cancer are largely limited by the extent of tumor invasion and the presence of lymph node and distant metastases. Therefore, exploring the biological behavior of pancreatic cancer cells (PCCs) is extremely important for the understanding, diagnosis, and treatment of pancreatic cancer. Current studies have shown that pancreatic stellate cells (PSCs) regulate the biological behavior of PCCs, such as their proliferation, apoptosis, invasion, and migration, by remodeling the extracellular matrix. Though Hic-5 is an important gene in PSCs, no study has investigated the regulation of PCCs by Hic-5. Here, we demonstrate that Hic-5 expression is upregulated in pancreatic cancer and that siRNA transfection can effectively inhibit Hic-5 expression. Compared to the control group, Hic-5 inhibition significantly reduced proliferation, increased apoptosis, and reduced invasion and migration of PCCs. Moreover, the inhibition of Hic-5 expression simultaneously reduced matrix metalloproteinase-9 (MMP-9) expression. Statistical analysis revealed that Hic-5 expression was higher among the pancreatic cancer group than among the normal group and was negatively correlated with postoperative survival time among patients with pancreatic cancer. These results have important clinical significance for further exploring the molecular mechanism involved in Hic-5-mediated invasion and metastasis of pancreatic cancer and ameliorating the prognosis of patients with pancreatic cancer.

A mitochondrial ROS pathway controls matrix metalloproteinase 9 levels and invasive properties in RAS-activated cancer cells.

Matrix metalloproteinases (MMPs) are tissue-remodeling enzymes involved in the processing of various biological molecules. MMPs also play important roles in cancer metastasis, contributing to angiogenesis, intravasation of tumor cells, and cell migration and invasion. Accordingly, unraveling the signaling pathways controlling MMP activities could shed additional light on cancer biology. Here, we report a molecular axis, comprising the molecular adaptor hydrogen peroxide-inducible clone-5 (HIC-5), NADPH oxidase 4 (NOX4), and mitochondria-associated reactive oxygen species (mtROS), that regulates MMP9 expression and may be a target to suppress cancer metastasis. We found that this axis primarily downregulates mtROS levels which stabilize MMP9 mRNA. Specifically, HIC-5 suppressed the expression of NOX4, the source of the mtROS, thereby decreasing mtROS levels and, consequently, destabilizing MMP9 mRNA. Interestingly, among six cancer cell lines, only EJ-1 and MDA-MB-231 cells exhibited upregulation of NOX4 and MMP9 expression after shRNA-mediated HIC-5 knockdown. In these two cell lines, activating RAS mutations commonly occur, suggesting that the HIC-5-mediated suppression of NOX4 depends on RAS signaling, a hypothesis that was supported experimentally by the introduction of activated RAS into mammary epithelial cells. Notably, HIC-5 knockdown promoted lung metastasis of MDA-MB-231 cancer cells in mice. The tumor growth of HIC-5-silenced MDA-MB-231 cells at the primary sites was comparable to that of control cells. Consistently, the invasive properties of the cells, but not their proliferation, were enhanced by the HIC-5 knockdown in vitro. We conclude that NOX4-mediated mtROS signaling increases MMP9 mRNA stability and affects cancer invasiveness but not tumor growth.

RETRACTED: Astragaloside IV reduces the hypoxia-induced injury in PC-12 cells by inhibiting expression of miR-124.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article "… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

The adaptor protein ARA55 and the nuclear kinase HIPK1 assist c-Myb in recruiting p300 to chromatin.

LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin.

Hic-5 regulates epithelial to mesenchymal transition in ovarian cancer cells in a TGFß1-independent manner.

The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. We have previously identified the hydrogen peroxide-inducible clone-5 (Hic-5) gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. Hic-5 is a focal adhesion scaffold protein and has been primarily studied for its role as a key mediator of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) in epithelial cells of both normal and malignant origin; however, its role in EOC has been never investigated. Here we demonstrate that Hic-5 is overexpressed in advanced EOC, and that Hic-5 is upregulated upon TGFß1 treatment in the EOC cell line with epithelial morphology (A2780s), associated with EMT induction. However, ectopic expression of Hic-5 in A2780s cells induces EMT independently of TGFß1, accompanied with enhancement of cellular proliferation rate and migratory/invasive capacity and increased resistance to chemotherapeutic drugs. Moreover, Hic-5 knockdown in the EOC cells with mesenchymal morphology (SKOV3) was accompanied by induction of mesenchymal-to-epithelial transition (MET), followed by a reduction of their proliferative, migratory/invasive capacity, and increased drugs sensitivity in vitro, as well as enhanced tumor cell colonization and metastatic growth in vivo. The modulation of Hic-5 expression in EOC cells resulted in altered regulation of numerous EMT-related canonical pathways and was indicative for a possible role of Hic-5 in controlling EMT through a RhoA/ROCK mediated mechanism. To our knowledge, this is the first report examining the role of Hic-5 in EOC, and its role in maintaining the mesenchymal phenotype of EOC cells independently of exogenous TGFß1 treatment.

Identification of Hic-5 as a novel scaffold for the MKK4/p54 JNK pathway in the development of abdominal aortic aneurysms.

BACKGROUND: Although increased amounts of reactive oxygen species in the pathogenesis of abdominal aortic aneurysm (AAA) are well documented, the precise molecular mechanisms by which reactive oxygen species induce AAAs have not been fully elucidated. This study focused on the role of hydrogen peroxide-inducible clone 5 (Hic-5), which is induced by hydrogen peroxide and transforming growth factor-ß, in the cellular signaling of AAA pathogenesis. METHODS AND RESULTS: Using the angiotensin II-induced AAA model in Apoe(-/-) mice, we showed that Apoe(-/-)Hic-5(-/-) mice were completely protected from AAA formation and aortic rupture, whereas Apoe(-/-) mice were not. These features were similarly observed in smooth muscle cell-specific Hic-5-deficient mice. Furthermore, angiotensin II treatment induced Hic-5 expression in a reactive oxygen species-dependent manner in aortic smooth muscle cells in the early stage of AAA development. Mechanistic studies revealed that Hic-5 interacted specifically with c-Jun N-terminal kinase p54 and its upstream regulatory molecule mitogen-activated protein kinase kinase 4 as a novel scaffold protein, resulting in the expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase 2 activation in aortic smooth muscle cells. CONCLUSION: Hic-5 serves as a novel scaffold protein that specifically activates the mitogen-activated protein kinase kinase 4/p54 c-Jun N-terminal kinase pathway, thereby leading to the induction and activation of matrix metalloproteinases in smooth muscle cells and subsequent AAA formation. Our study provided a novel therapeutic option aimed at inhibiting the mitogen-activated protein kinase kinase 4-Hic-5-p54 c-Jun N-terminal kinase pathway in the vessel wall, particularly through Hic-5 inhibition, which may be used to produce more precise and effective therapies.