Receptor-interacting Protein Kinase 4 Research Articles

GSK3β and UCHL3 govern RIPK4 homeostasis via deubiquitination to enhance tumor metastasis in ovarian cancer.

Receptor-interacting protein kinase 4 (RIPK4) is increasingly recognized as a pivotal player in ovarian cancer, promoting tumorigenesis and disease progression. Despite its significance, the posttranslational modifications dictating RIPK4 stability in ovarian cancer remain largely uncharted. In this study, we first established that RIPK4 levels are markedly higher in metastatic than in primary ovarian cancer tissues through single-cell sequencing. Subsequently, we identified UCHL3 as a key deubiquitinase that regulates RIPK4. We elucidate the mechanism that UCHL3 interacts with and deubiquitinates RIPK4 at the K469 site, removing the K48-linked ubiquitin chain and thus enhancing RIPK4 stabilization. Intriguingly, inhibition of UCHL3 activity using TCID leads to increased RIPK4 ubiquitination and degradation. Furthermore, we discovered that GSK3β-mediated phosphorylation of RIPK4 at Ser420 enhances its interaction with UCHL3, facilitating further deubiquitination and stabilization. Functionally, RIPK4 was found to drive the proliferation and metastasis of ovarian cancer in a UCHL3-dependent manner both in vitro and in vivo. Importantly, positive correlations between RIPK4 and UCHL3 protein expression levels were observed, with both serving as indicators of poor prognosis in ovarian cancer patients. Overall, this study uncovers a novel pathway wherein GSK3β-induced phosphorylation of RIPK4 strengthens its interaction with UCHL3, leading to increased deubiquitination and stabilization of RIPK4, thereby promoting ovarian cancer metastasis. These findings offer new insights into the molecular underpinnings of ovarian cancer and highlight potential therapeutic targets for enhancing antitumor efficacy.

RIPK4 Promotes Cell Invasion and the Epithelial-Mesenchymal Transition in Ovarian Cancer.

To investigate the clinical role and biological function of receptor-interacting protein kinase 4 (RIPK4) in ovarian cancer (OC). We conducted a comprehensive analysis of the expression and prognostic role of RIPK4 in OC using various public databases including The Cancer Genome Atlas, Oncomine, and Kaplan-Meier plotter. In vitro studies included wound healing, cell migration and invasion, cell proliferation, and cell apoptosis assays as well as vascular mimicry experiments. In vivo studies were conducted using subcutaneous and intraperitoneal xenografts. Our findings revealed that RIPK4 was significantly overexpressed in OC tissue compared to normal ovarian tissue. Moreover, the overexpression of RIPK4 was associated with advanced-stage disease and a poor prognosis in OC patients. RIPK4 silencing resulted in significant inhibition of intraperitoneal tumor growth, invasion, and vascular mimicry in OC cells. Furthermore, downregulation of RIPK4 inhibited the epithelial-mesenchymal transition of OC cells both in vitro and in vivo by promoting the expression of E-cadherin and inhibiting the expression of N-cadherin. The results of this study suggest that RIPK4 may function as an oncogene in the development and prognosis of OC.

RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Depletion of RIPK4 parallels higher malignancy potential in cutaneous squamous cell carcinoma.

BackgroundThe RIPK4 (receptor-interacting protein kinase 4), a member of the RIPK family, acts as an important regulator of epidermal differentiation, cutaneous inflammation, and cutaneous wound repair. However, Until now, the role of RIPK4 in tumorigenesis remains elusive. There have been no studies exploring the effects of RIPK4 on the signaling pathway in cutaneous squamous cell carcinoma (SCC). It remains unknown whether RIPK4 expression, which can affect the degree of epidermal differentiation can also influence the radiosensitivity of skin SCC. It is urgent to fully elucidate the biological mechanism by which RIPK4 promotes carcinogenesis in skin SCC and determine whether RIPK4 expression levels predicts the sensitivity to radiotherapy in skin SCC. MethodsHuman skin SCC cell line, A431, was transfected with either small interfering RNAs (siRNAs) targeting RIPK4 (siR-RIPK4) or negative control siRNA (siR-NC). Western blotting was used to detect the expression of RIPK4 and Raf/MEK/ERK pathway-related proteins. The cells were irradiated using an X-ray irradiator at 6 MV with different radiation doses (0, 2, 6, and 10 Gy). Cell proliferation analysis, colony formation assay, transwell cell migration and invasion assay, cell cycle and apoptosis analysis were conducted to investigate the effect of RIPK4 silencing on skin SCC malignancy and radiosensitivity.ResultsRIPK4 protein expression was significantly decreased in the A431 cells transfected with siR-RIPK4, compared with the A431 cells transfected with siR-NC. RIPK4 silencing facilitated the proliferation, colony formation, migration, and invasion ability of A431 cell line, while cell cycle progression or cell apoptosis were not significantly influenced. In contrast with the previous literature, Raf/MEK/ERK pathway was not effected by RIPK4 knockdown in skin SCC. RIPK4 knockdown could not reverse the radiation resistance of A431 cells to irradiation in vitro.ConclusionsIn general, although depletion of RIPK4 cannot reverse the radiation resistance of A431 cells in vitro, it parallels higher malignancy potential in cutaneous SCC. To our knowledge, this is the first report of the effects of RIPK4 expression on the Raf/MEK/ERK signaling pathway and radiosensitivity in cutaneous SCC. The better understanding of the molecular mechanism of RIPK4 in cutaneous SCC may provide a promising biomarker for skin SCC prognosis and treatment.

Deciphering the Functional Role of RIPK4 in Melanoma.

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4high) or independent (RIPK4low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

Receptor interacting protein kinase 4 promotes cell proliferation, migration, and invasion in ovarian cancer via targeting protein kinase C delta

Receptor interacting protein kinase 4 (RIPK4) has been reported to function as an oncogenic role in several types of cancers. The aim of this study was to evaluate the role of RIPK4 in ovarian cancer (OC) cells and to elucidate the mechanism behind this effect. In this study, the GEPIA database was used to analyze the RIPK4 expressions in OC tissues and overall survival. qRT-PCR and western blot assay were performed to detect the expressions of RIPK4 and protein kinase C delta (PRKCD) in OC cells. In addition, cell proliferation was assessed by CCK-8 and colony formation assay while cell invasion and migration were evaluated by transwell, wound healing and western blot assay. The interaction of RIPK4 and PRKCD was analyzed by the STRING database and the bioGRID database, and verified with co-immunoprecipitation. Herein, we describe that RIPK4 expression was upregulated in OC tissues and cells and was associated with poor overall survival. RIPK4 silencing repressed the proliferation, migration, and invasion of OC cells. Mechanistically, PRKCD was highly expressed in OC cells and was combined with RIPK4. PRKCD was highly positively associated with RIPK4 in OC and was regulated by RIPK4. Moreover, PRKCD overexpression reversed the inhibitory effects of RIPK4 silencing on OC cell proliferation, migration, and invasion. RIPK4 functions as an oncogene in OC cells via at least partially binding to PRKCD, which might represent a novel therapeutic strategy for improving survival for patients with OC.

Overexpression of RIPK4 Predicts Poor Prognosis and Promotes Metastasis in Ovarian Cancer.

This study was conducted to evaluate the prognostic value of receptor-interacting protein kinase 4 (RIPK4) in ovarian cancer (OC) and its role in tumorigenesis. RNA expression and the corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The relationship between clinical-pathological characteristics and RIPK4 expression was analyzed using the Wilcoxon signed-rank test and logistic regression. The Cox regression and the Kaplan-Meier method were used to evaluate the relationship between clinicopathological features and overall survival (OS). Gene set enrichment analysis (GSEA) was performed using Molecular Signatures Database. Scratch assay, transwell assay, and cell transfection were used to verify the function of RIPK4. Overexpression of RIPK4 was associated with the stage of OC and distant metastasis. Survival analysis revealed that patients with OC and higher expression of RIPK4 had a poorer prognosis. Univariate and multivariate analyses indicated that high expression of RIPK4 was associated with poor OS, as well as age and stage of OC. The areas under the curve (AUC) at 1, 4, and 8 years were 0.737, 0.634, and 0.669, respectively, according to the established OS prediction model. GSEA revealed that adherens junction, cadherin binding, and Wnt signaling pathway were enriched in the high RIPK4 expression group. Cell transfection confirmed RIPK4 was involved in the Wnt signaling pathway. RIPK4 can act as a potential prognostic molecular marker for poor survival in OC. Moreover, RIPK4 is associated with tumor metastasis and implicated in the regulation of the Wnt signaling pathway.

Insight Into the Function of RIPK4 in Keratinocyte Differentiation and Carcinogenesis.

The receptor-interacting protein kinase 4 (RIPK4), a member of the RIPK family, was originally described as an interaction partner of protein kinase C (PKC) β and PKCδ. RIPK4 is identified as a key regulator of keratinocyte differentiation, cutaneous inflammation, and cutaneous wound repair. The mechanism by which RIPK4 integrates upstream signals to initiate specific responses remains elusive. Previous studies have indicated that RIPK4 can regulate several signaling pathways, including the NF-κB, Wnt/β-catenin, and RAF/MEK/ERK pathways. Furthermore, RIPK4-related biological signaling pathways interact with each other to form a complex network. Mounting evidence suggests that RIPK4 is aberrantly expressed in various kinds of cancers. In several types of squamous cell carcinoma (SCC), the mutations that drive aggressive SCC have been found in RIPK4. In addition, the function of RIPK4 in carcinogenesis is probably tissue-specific, since RIPK4 can play a dual role as both a tumor promoter and a tumor suppressor in different tumor types. Therefore, RIPK4 may represent as an independent prognostic factor and a promising novel therapeutic target, which can be used to identify the risks of patients and guide personalized treatments. In future, RIPK4-interacting pathways and precise molecular targets need to be investigated in order to further elucidate the mechanisms underlying epidermal differentiation and carcinogenesis.

Silencing of RIPK4 inhibits epithelial‑mesenchymal transition by inactivating the Wnt/β‑catenin signaling pathway in osteosarcoma.

Receptor interacting protein kinase 4 (RIPK4) is a serine/threonine kinase that plays an important role in the regulation of cell proliferation, invasion and metastasis in several malignancies; however, its clinical significance and biological function in osteosarcoma (OS) remains unknown. In the present study, the RIPK4 expression level was significantly upregulated in OS tissues and cell lines. High RIPK4 expression was positively associated with larger sized tumors, advanced Enneking stage and poor prognosis in patients with OS. Furthermore, the results revealed that RIPK4 knockdown in the OS cell lines MG-63 and U2OS reduced cell migration and invasion via the inhibition of epithelial-mesenchymal transition (EMT) process, whereby E-cadherin expression was increased and N-cadherin and vimentin expression decreased. Mechanistically, RIPK4 knockdown inhibited EMT by inactivating the Wnt/β-catenin signaling pathway. These findings suggest that RIPK4 may be a novel potential therapeutic target for the treatment of metastases in patients with OS.

Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4.

The epidermis, the outer layer of the skin, is formed by stratified keratinocyte layers. The self-renewal of the epidermis is provided by sustained proliferation and differentiation of the keratinocyte stem cells localized to the basal layer of the epidermis. Receptor-interacting protein kinase 4 (RIPK4) is an important regulator of keratinocyte differentiation, mutations of which are associated with congenital ectodermal malformations. In an attempt to identify the molecular basis of RIPK4’s function, we applied yeast two-hybrid screen (Y2H) and found basal layer-specific keratin filament component keratin 14 (KRT14) as a novel RIPK4-interacting partner. During keratinocyte differentiation, layer-specific keratin composition is tightly regulated. Likewise, the basal layer specific KRT14/keratin 5 (KRT5) heterodimers are replaced by keratin 1 (KRT1)/keratin 10 (KRT10) in suprabasal layers. The regulation of keratin turnover is under the control of signaling associated with posttranslational modifications in which phosphorylation plays a major role. In this study, we verified the KRT14-RIPK4 interaction, which was identified with Y2H, in mammalian cells and showed that the interaction was direct by using proteins expressed in bacteria. According to our results, the N-terminal kinase domain of RIPK4 is responsible for KRT14-RIPK4 interaction; however, the RIPK4 kinase activity is dispensable for the interaction. In accordance with their interaction, RIPK4 and KRT14 colocalize within the cells, particularly at keratin filaments associated with perinuclear ring-like structures. Moreover, RIPK4 did not show any effect on KRT14/KRT5 heterodimer formation. Our results suggest that RIPK4 may regulate the keratin turnover required for keratinocyte differentiation through interacting with KRT14.

RIP4 upregulates CCL20 expression through STAT3 signalling in cultured keratinocytes.

The receptor-interacting protein kinase 4 (RIP4), a serine/threonine kinase, is an important modulator of epidermal growth and cutaneous inflammation. We found that RIP4 expression was significantly increased in the lesional skin of psoriasis. However, the role and regulatory mechanism of RIP4 in psoriasis have not been characterized. After treatment with IL-17, RIP4 mRNA and protein levels were increased in HaCaT cells. IL-17 also activated the RIP4 promoter. To understand the functional role of RIP4 in keratinocyte and to investigate the genes regulated by RIP4, RNA-based microarray analysis was performed. Among immune response-related genes, CCL20 expression was significantly changed by RIP4. To identify RIP4-interacting protein, an immunoprecipitation assay was performed. As a result, STAT3 was identified as a new protein that interacts with RIP4. The interaction of RIP4 and STAT3 enhanced STAT3 phosphorylation. In addition, the transcriptional activity of STAT3 induced by RIP4 regulated IL-17-mediated CCL20 expression in HaCaT cells. Taken together, these findings indicate that IL-17 increased RIP4-mediated STAT3 phosphorylation by directly interacting with STAT3. Thus, transcriptional activation of STAT3 promotes the expression of CCL20. Thus, activations of these signalling pathways by RIP4 may contribute to epithermal inflammation in psoriatic keratinocytes.

RIPK4 promotes bladder urothelial carcinoma cell aggressiveness by upregulating VEGF-A through the NF-\u03baB pathway

BackgroundConstitutively activated nuclear factor kappa B (NF-κB) signalling plays vital roles in bladder urothelial carcinoma (BC) progression. We investigate the effect of receptor-interacting protein kinase 4 (RIPK4) on NF-κB activation and BC progression.MethodsThe expression of RIPK4 was examined in 25 cryopreserved paired bladder samples and 112 paraffin BC specimens. In vivo and in vitro assays were performed to validate effect of RIPK4 on NF-κB pathway-mediated BC progression.ResultsHigh expression of RIPK4 was observed in BC tissues and was an independent predictor for poor overall survival. Up or downregulating the expression of RIPK4 enhanced or inhibited, respectively, the migration and invasion of BC cells in vitro and in vivo. Mechanistically, RIPK4 promoted K63-linked polyubiquitination of tumour necrosis factor receptor-associated factor 2 (TRAF2), receptor-interacting protein (RIP) and NF-κB essential modulator (NEMO). RIPK4 also promoted nuclear localisation of NF-κB-p65, and maintained activation of NF-κB substantially, leading to upregulation of VEGF-A, ultimately promoting BC cell aggressiveness.ConclusionsOur data highlighted the molecular aetiology and clinical significance of RIPK4 in BC: upregulation of RIPK4 contributes to NF-κB activation, and upregulates VEGF-A, and BC progression. Targeting RIPK4 might represent a new therapeutic strategy to improve survival for patients with BC.

A20 regulates canonical wnt-signaling through an interaction with RIPK4.

A20 is a ubiquitin-editing enzyme that is known to regulate inflammatory signaling and cell death. However, A20 mutations are also frequently found in multiple malignancies suggesting a potential role as a tumor suppressor as well. We recently described a novel role for A20 in regulating the wnt-beta-catenin signaling pathway and suppressing colonic tumor development in mice. The underlying mechanisms for this phenomenon are unclear. To study this, we first generated A20 knockout cell lines by genome-editing techniques. Using these cells, we show that loss of A20 causes dysregulation of wnt-dependent gene expression by RNAseq. Mechanistically, A20 interacts with a proximal signaling component of the wnt-signaling pathway, receptor interacting protein kinase 4 (RIPK4), and regulation of wnt-signaling by A20 occurs through RIPK4. Finally, similar to the mechanism by which A20 regulates other members of the receptor interacting protein kinase family, A20 modifies ubiquitin chains on RIPK4 suggesting a possible molecular mechanism for A20’s control over the wnt-signaling pathway.

Crystal Structure of Ripk4 Reveals Dimerization-Dependent Kinase Activity

Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities. We describe the structure of the murine Ripk4 (MmRipk4) kinase domain, in ATP- and inhibitor-bound forms. The crystallographic dimer of MmRipk4 is similar to those of RIPK2 and BRAF, and we show that the intact dimeric entity is required for MmRipk4 catalytic activity through a series of engineered mutations and cell-based assays. We also assess the impact of BPS mutations on protein structure and activity to elucidate the molecular origins of the disease.

P124 THE UBIQUITIN EDITING ENZYME A20 LINKS INFLAMMATION AND COLITIS ASSOCIATED CANCER

Inflammatory bowel disease (IBD) patients are at increased risk of colorectal cancer (CRC)1. Tumor initiating events in colitis-associated cancers (CACs) differ significantly from sporadic CRC2. In CACs, hyperactivity of wnt/beta-catenin signaling typically occur without adenomatous polyposis coli gene mutations commonly seen in sporadic colon cancers. This suggests distinct mechanisms cause dysregulation of wnt signaling in CACs. A20 (TNFAIP3) is a ubiquitin-editing enzyme, linked to IBD, with roles in dampening inflammation3. We found that A20 is a tumor suppressor in the murine colon directly inhibiting beta-catenin signaling4. We now report regulation of wnt signaling by A20 depends on interaction with receptor interacting protein kinase 4 (RIPK4). Interestingly, A20’s function downstream of inflammatory signaling depends upon the closely related proteins RIPK1 and RIPK2 suggesting a conserved interaction between A20 and this family of proteins. Using a mammalian yeast-2-hybrid system we found A20 and RIPK4 interact (Fig. 1A). This association was confirmed via immunoprecipitation using endogenous proteins and was dependent upon wnt stimulation (Fig. 1B). Using a beta-catenin luciferase reporter assay, we showed siRNA knockdown of RIPK4 partially abrogates enhanced wnt signaling in absence of A20 (Fig. 1C), suggesting A20 negatively regulates wnt signaling via RIPK4. Mechanistically, we found A20 normally supports K48-linked ubiquitination of RIPK4 (Fig. 1D). We then determined whether A20 plays a role in regulating CACs with the AOM-DSS model of CAC in mice with a targeted deletion of A20 in the intestinal epithelium. Preliminary data suggests A20FL/FL Villin-Cre mice treated with AOM-DSS develop larger tumors than control mice (Figs. 2A,2B). Overall, we propose that A20 restricts CACs by inhibiting the activity of RIPK4. 1. Gut. 2001;48(4):526–35. 2. Cancer Res. 1995;55(10):2035–38. 3. Nat. Rev. Immunol. 2012;12:774–85. 4. PLoS One. 2013;8(5):e62223.

Keratinocyte-Specific Ablation of RIPK4 Allows Epidermal Cornification but Impairs Skin Barrier Formation

In humans, receptor-interacting protein kinase 4 (RIPK4) mutations can lead to the autosomal recessive Bartsocas-Papas and popliteal pterygium syndromes, which are characterized by severe skin defects, pterygia, as well as clefting. We show here that the epithelial fusions observed in RIPK4 full knockout (KO) mice are E-cadherin dependent, as keratinocyte-specific deletion of E-cadherin in RIPK4 full KO mice rescued the tail-to-body fusion and fusion of oral epithelia. To elucidate RIPK4 function in epidermal differentiation and development, we generated epidermis-specific RIPK4 KO mice (RIPK4EKO). In contrast to RIPK4 full KO epidermis, RIPK4EKO epidermis was normally stratified and the outside-in skin barrier in RIPK4EKO mice was largely intact at the trunk, in contrast to the skin covering the head and the outer end of the extremities. However, RIPK4EKO mice die shortly after birth due to excessive water loss because of loss of tight junction protein claudin-1 localization at the cell membrane, which results in tight junction leakiness. In contrast, mice with keratinocyte-specific RIPK4 deletion during adult life remain viable. Furthermore, our data indicate that epidermis-specific deletion of RIPK4 results in delayed keratinization and stratum corneum maturation and altered lipid organization and is thus indispensable during embryonic development for the formation of a functional inside-out epidermal barrier.

P124 THE UBIQUITIN EDITING ENZYME A20 LINKS INFLAMMATION AND COLITIS ASSOCIATED CANCER

Inflammatory bowel disease (IBD) patients are at increased risk of colorectal cancer (CRC)1. Tumor initiating events in colitis-associated cancers (CACs) differ significantly from sporadic CRC2. In CACs, hyperactivity of wnt/beta-catenin signaling typically occur without adenomatous polyposis coli gene mutations commonly seen in sporadic colon cancers. This suggests distinct mechanisms cause dysregulation of wnt signaling in CACs. A20 (TNFAIP3) is a ubiquitin-editing enzyme, linked to IBD, with roles in dampening inflammation3. We found that A20 is a tumor suppressor in the murine colon directly inhibiting beta-catenin signaling4. We now report regulation of wnt signaling by A20 depends on interaction with receptor interacting protein kinase 4 (RIPK4). Interestingly, A20’s function downstream of inflammatory signaling depends upon the closely related proteins RIPK1 and RIPK2 suggesting a conserved interaction between A20 and this family of proteins. Using a mammalian yeast-2-hybrid system we found A20 and RIPK4 interact (Fig. 1A). This association was confirmed via immunoprecipitation using endogenous proteins and was dependent upon wnt stimulation (Fig. 1B). Using a beta-catenin luciferase reporter assay, we showed siRNA knockdown of RIPK4 partially abrogates enhanced wnt signaling in absence of A20 (Fig. 1C), suggesting A20 negatively regulates wnt signaling via RIPK4. Mechanistically, we found A20 normally supports K48-linked ubiquitination of RIPK4 (Fig. 1D). We then determined whether A20 plays a role in regulating CACs with the AOM-DSS model of CAC in mice with a targeted deletion of A20 in the intestinal epithelium. Preliminary data suggests A20FL/FL Villin-Cre mice treated with AOM-DSS develop larger tumors than control mice (Figs. 2A,2B). Overall, we propose that A20 restricts CACs by inhibiting the activity of RIPK4. 1. Gut. 2001;48(4):526-35. 2. Cancer Res. 1995;55(10):2035-38. 3. Nat. Rev. Immunol. 2012;12:774-85. 4. PLoS One. 2013;8(5):e62223.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Influence of protein kinase RIPK4 expression on the apoptosis and proliferation of chondrocytes in osteoarthritis.

The present study aimed to investigate the expression of receptor-interacting protein kinase 4 (RIPK4) and its effect on the apoptosis and proliferation of chondrocytes in osteoarthritis (OA). A total of 28 OA cartilage tissues and 20 normal cartilage tissues were collected to detect the expression of RIPK4 by using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Chondrocytes were isolated from OA cartilage tissues and divided into OA, NC, si-RIPK4, Wnt3a, and si-RIPK4+Wnt3a groups, and those isolated from normal cartilage tissues were considered the Normal group. Chondrocytes proliferation was detected by MTT assay, cell apoptosis was indicated using flow cytometry and Wnt/β-catenin signaling pathway related-proteins were investigated using western blot analysis. RIPK4 mRNA and protein expression levels in OA cartilage tissues and OA chondrocytes were increased compared with normal controls (all P<0.05). Additionally, OA chondrocytes showed reduced cell proliferation, increased cell apoptosis and upregulated expression levels of Wnt/β-catenin signaling pathway related-proteins (all P<0.05). Once transfected with si-RIPK4, the proliferation ability of chondrocytes was enhanced, but apoptosis was notably decreased. Furthermore, the expression levels of Wnt/β-catenin signaling pathway related-proteins were significantly downregulated (all P<0.05). Results indicated that Wnt3a reversed the effect of si-RIPK4 on chondrocyte proliferation and apoptosis (all P<0.05). Thus, silencing RIPK4 promoted the proliferation and inhibited the apoptosis of chondrocytes. In addition, silencing RIPK4 blocked the Wnt/β-catenin signaling pathway, thus contributing to alleviating the OA pathogenesis.

Interferon Regulatory Factor 6 Promotes Keratinocyte Differentiation in Response to Porphyromonas gingivalis.

We recently demonstrated that the expression of the interferon regulatory factor 6 (IRF6) transcription factor in oral keratinocytes was stimulated by the periodontal pathogen Porphyromonas gingivalis Here, we have established that IRF6 promotes the differentiation of oral keratinocytes in response to P. gingivalis This was evidenced by the IRF6-dependent upregulation of specific markers of keratinocyte terminal differentiation (e.g., involucrin [IVL] and keratin 13 [KRT13]), together with additional transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 (GRHL3) and Ovo-like zinc finger 1 (OVOL1). We have previously established that the transactivator function of IRF6 is activated by receptor-interacting protein kinase 4 (RIPK4). Consistently, the silencing of RIPK4 inhibited the stimulation of IVL, KRT13, GRHL3, and OVOL1 gene expression. IRF6 was shown to also regulate the stimulation of transglutaminase-1 (TGM1) gene expression by P. gingivalis, as well as that of small proline-rich proteins (e.g., SPRR1), which are covalently cross-linked by TGM1 to other proteins, including IVL, during cornification. The expression of the tight junction protein occludin (OCLN) was found to also be upregulated in an IRF6-dependent manner. IRF6 was demonstrated to be important for the barrier function of oral keratinocytes; specifically, silencing of IRF6 increased P. gingivalis-induced intercellular permeability and cell invasion. Taken together, our findings potentially position IRF6 as an important mediator of barrier defense against P. gingivalis.

RIPK4 activates an IRF6-mediated proinflammatory cytokine response in keratinocytes

Keratinocytes of the oral mucosa and epidermis play key roles in host defense. In addition to functioning as a physical barrier, they also produce cytokines to elicit inflammation in response to infection or injury. We recently established that receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) function as a cell-intrinsic signaling axis to regulate keratinocyte differentiation. In this study, we have demonstrated a functional relationship between RIPK4 and IRF6 in the control of proinflammatory cytokine expression in keratinocytes. The overexpression of RIPK4 by oral keratinocytes induced the strong expression of CCL5 and CXCL11. In contrast, the expression of other cytokines (e.g. IL8 and TNF) was largely unaffected, thus demonstrating specificity in the induction of proinflammatory cytokine expression by RIPK4. CCL5 and CXCL11 expression were also induced in response to the activation of the PKC pathway, and gene silencing experiments indicated that their inducible expression was dependent on RIPK4 and IRF6. Moreover, gene reporter assays suggested that RIPK4 induces CCL5 and CXCL11 expression by stimulating the transactivation of their promoters by IRF6. Accordingly, our findings suggest that the RIPK4-IRF6 signaling axis plays a multifaceted role in barrier epithelial homeostasis through its regulation of both keratinocyte inflammation and differentiation.