ABSTRACT
The TNF superfamily ligands APRIL and BAFF bind with different affinity to two receptors, BCMA and TACI, and induce cell survival and/or proliferation, whereas BAFF also binds specifically to BAFFR. These molecules were considered specific for the immune system. Recently, however, they were also found in epithelial and mesenchymal noncancerous and cancerous tissues and cell lines. In this article, we report that hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B and HCC specimens express APRIL and BAFF and their receptors BCMA and BAFFR, but not TACI; APRIL/BCMA is enhanced in HCC, compared with normal liver tissue. In contrast to previous reports, APRIL binding to BCMA decreases cell proliferation by inducing G(2)/M cell cycle arrest, whereas BAFF has no effect on cell growth. HCC cells therefore represent a rare system in which these two ligands (APRIL and BAFF) exert a differential effect and may serve as a model for specific APRIL/BCMA actions. We show that the effect of APRIL is mediated via BCMA, which does not activate the classical NF-κB pathway, whereas it induces a novel signaling pathway, which involves JNK2 phosphorylation, FOXO3A activation, and GADD45 transcription. In addition, JNK2 mediates the phosphorylation of Akt, which is activated but does not participate in the antiproliferative effect of APRIL. Furthermore, transcriptome analysis revealed that APRIL modifies genes specifically related to cell cycle modulation, including MCM2/4/5/6, CDC6, PCNA, and POLE2. Our data, therefore, identify a novel APRIL/BCMA signaling pathway in HCC and suggest that APRIL could have a pleiotropic role in tumor biology.
Subject(s)
B-Cell Maturation Antigen/immunology , Cell Cycle Proteins/immunology , DNA-Binding Proteins/immunology , Forkhead Transcription Factors/immunology , G2 Phase Cell Cycle Checkpoints/immunology , Liver/immunology , M Phase Cell Cycle Checkpoints/immunology , MAP Kinase Kinase 7/immunology , Nuclear Proteins/immunology , Transcription Factors/immunology , B-Cell Activating Factor/genetics , B-Cell Activating Factor/immunology , B-Cell Activating Factor/metabolism , B-Cell Maturation Antigen/genetics , B-Cell Maturation Antigen/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , G2 Phase Cell Cycle Checkpoints/genetics , Hep G2 Cells , Humans , Liver/cytology , M Phase Cell Cycle Checkpoints/genetics , MAP Kinase Kinase 7/genetics , MAP Kinase Kinase 7/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation/genetics , Phosphorylation/immunology , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/immunology , Proliferating Cell Nuclear Antigen/metabolism , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/genetics , Transcription, Genetic/immunologyABSTRACT
The soluble TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) binds to the fibroblast growth factor-inducible 14 receptor (FN14, TNFRSF12A) on the cell membrane and induces multiple biological responses, such as proliferation, migration, differentiation, angiogenesis and apoptosis. Previous reports show that TWEAK, which does not contain a death domain in its cytoplasmic tail, induces the apoptosis of tumor cell lines through the induction of TNFα secretion. TWEAK induces apoptosis in human keratinocytes. Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins. The use of specific inhibitors and the absence of procaspase-3 cleavage suggest that the apoptosis of keratinocytes follows a caspase- and cathepsin B-independent pathway. Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes. Confocal microscopy showed that TWEAK induces the cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, thus initiating caspase-independent apoptosis. Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase. Finally, we report that TWEAK and FN14 are normally expressed in the basal layer of the physiological epidermis and are greatly enhanced in benign (psoriasis) and malignant (squamous cell carcinoma) skin pathologies that are characterized by an inflammatory component. TWEAK might play an essential role in skin homeostasis and pathology.
Subject(s)
Apoptosis Inducing Factor/metabolism , Apoptosis/physiology , G2 Phase Cell Cycle Checkpoints/physiology , Keratinocytes/cytology , Keratinocytes/metabolism , Tumor Necrosis Factors/metabolism , Active Transport, Cell Nucleus , CDC2 Protein Kinase , Caspases/metabolism , Cathepsin B/metabolism , Cell Line , Cyclin B/metabolism , Cyclin B1/metabolism , Cyclin-Dependent Kinases , Cytokine TWEAK , Humans , Inflammation/metabolism , Inflammation/pathology , Psoriasis/metabolism , Psoriasis/pathology , Receptors, Tumor Necrosis Factor/metabolism , Skin/cytology , Skin/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , TNF-Related Apoptosis-Inducing Ligand/biosynthesis , TWEAK Receptor , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
TNFα is known to be expressed in human skin, regulating immune-related responses. Here we report that human normal skin keratinocytes express the members of the TNF superfamily members A proliferation-inducing ligand (APRIL; TNFSF13), B cell-activating factor (BAFF; TNFSF13B), and their receptors, B cell maturation antigen (BCMA; TNFRSF17) and transmembrane activator, calcium-modulator, and cyclophilin ligand interactor (TACI; TNFRSF13B), in a distinct spatial pattern. Our data show a differential expression of these molecules within epidermal layers and skin appendages, whereas the BAFF-specific receptor BAFFR (TNFRSF13C) is absent. Importantly, APRIL and BCMA but not BAFF or TACI are up-regulated in inflammatory skin lesions of psoriasis and squamous cell carcinomas. To explore the functional significance of this system in the skin, we assayed these receptors and ligands in cultured primary keratinocytes and HaCaT cells. We show that both cell types express BAFF, APRIL, BCMA, and TACI. Furthermore, APRIL and/or BAFF trigger nuclear factor-κB activation and IL-6 and granulocyte macrophage colony-stimulating factor (GM-CSF) expression through functional BCMA receptors, an activation inhibited by anti-BCMA short hairpin RNA. However, BAFF and/or APRIL do not induce IL-8 or TNFα production. Our data advance BCMA as an inflammation-related TNFSFR member in keratinocytes, of potential importance in the management of inflammatory skin conditions.
