Persistent JunB activation in fibroblasts disrupts stem cell niche interactions enforcing skin aging.

Fibroblasts residing in the connective tissues constitute the stem cell niche, particularly in organs such as skin. Although the effect of fibroblasts on stem cell niches and organ aging is an emerging concept, the underlying mechanisms are largely unresolved. We report a mechanism of redox-dependent activation of transcription factor JunB, which, through concomitant upregulation of p16INK4A and repression of insulin growth factor-1 (IGF-1), initiates the installment of fibroblast senescence. Fibroblast senescence profoundly disrupts the metabolic and structural niche, and its essential interactions with different stem cells thus enforces depletion of stem cells pools and skin tissue decline. In fact, silencing of JunB in a fibroblast-niche-specific manner-by reinstatement of IGF-1 and p16 levels-restores skin stem cell pools and overall skin tissue integrity. Here, we report a role of JunB in the control of connective tissue niche and identified targets to combat skin aging and associated pathologies.

JUNB suppresses distant metastasis by influencing the initial metastatic stage.

The complex interactions between cells of the tumor microenvironment and cancer cells are considered a major determinant of cancer progression and metastasis. Yet, our understanding of the mechanisms of metastatic disease is not sufficient to successfully treat patients with advanced-stage cancer. JUNB is a member of the AP-1 transcription factor family shown to be frequently deregulated in human cancer and associated with invasion and metastasis. A strikingly high stromal JUNB expression in human breast cancer samples prompted us to functionally investigate the consequences of JUNB loss in cells of the tumor microenvironment on cancer progression and metastasis in mice. To adequately mimic the clinical situation, we applied a syngeneic spontaneous breast cancer metastasis model followed by primary tumor resection and identified stromal JUNB as a potent suppressor of distant metastasis. Comprehensive characterization of the JUNB-deficient tumor microenvironment revealed a strong influx of myeloid cells into primary breast tumors and lungs at early metastatic stage. In these infiltrating neutrophils, BV8 and MMP9, proteins promoting angiogenesis and tissue remodeling, were specifically upregulated in a JUNB-dependent manner. Taken together, we established stromal JUNB as a strong suppressor of distant metastasis. Consequently, therapeutic strategies targeting AP-1 should be carefully designed not to interfere with stromal JUNB expression as this may be detrimental for cancer patients.

JunB defines functional and structural integrity of the epidermo-pilosebaceous unit in the skin.

Transcription factors ensure skin homeostasis via tight regulation of distinct resident stem cells. Here we report that JunB, a member of the AP-1 transcription factor family, regulates epidermal stem cells and sebaceous glands through balancing proliferation and differentiation of progenitors and by suppressing lineage infidelity. JunB deficiency in basal progenitors results in a dermatitis-like syndrome resembling seborrheic dermatitis harboring structurally and functionally impaired sebaceous glands with a globally altered lipid profile. A fate switch occurs in a subset of JunB deficient epidermal progenitors during wound healing resulting in de novo formation of sebaceous glands. Dysregulated Notch signaling is identified to be causal for this phenotype. In fact, pharmacological inhibition of Notch signaling can efficiently restore the lineage drift, impaired epidermal differentiation and disrupted barrier function in JunB conditional knockout mice. These findings define an unprecedented role for JunB in epidermal-pilosebaceous stem cell homeostasis and its pathology.

Cytoplasmic localization of the cell polarity factor scribble supports liver tumor formation and tumor cell invasiveness.

The loss of epithelial cell polarity plays an important role in the development and progression of liver cancer. However, the specific molecular mechanisms supporting tumor initiation and progression are poorly understood. In this study, transcriptome data and immunofluorescence stains of tissue samples derived from hepatocellular carcinoma (HCC) patients revealed that overexpression associated with cytoplasmic localization of the basolateral cell polarity complex protein scribble (Scrib) correlated with poor prognosis of HCC patients. In comparison with HCC cells stably expressing wild-type Scrib (ScribWT ), mutated Scrib with enforced cytoplasmic enrichment (ScribP305L ) induced AKT signaling through the destabilization of phosphatase and tensin homolog (PTEN) and PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). Cytoplasmic ScribP305L stimulated a gene signature and a phenotype characteristic for epithelial to mesenchymal transition (EMT) and HCC cell invasiveness. ScribP305L -dependent invasion was mediated by the activator protein 1 (AP-1) constituents ATF2 and JunB through induction of paracrine-acting secreted protein acidic and cysteine-rich (SPARC). Coexpression of ScribP305L and the oncogene c-MYC through hydrodynamic gene delivery in mouse livers promoted tumor formation and increased abundance of pAKT, pATF2, and SPARC in comparison with controls. Finally, cytoplasmic Scrib localization correlated with AKT and ATF2 phosphorylation in human HCC tissues, and the ScribP305L -dependent gene signature was enriched in cancer patients with poor prognosis. CONCLUSION: Perturbation of hepatocellular polarity due to overexpression and cytoplasmic enrichment of Scrib supports tumor initiation and HCC cell dissemination through specific molecular mechanisms. Biomarker signatures identified in this study can be used for the identification of HCC patients with higher risk for the development of metastasis. (Hepatology 2018;67:1842-1856).

Junb controls lymphatic vascular development in zebrafish via miR-182.

JUNB, a subunit of the AP-1 transcription factor complex, mediates gene regulation in response to a plethora of extracellular stimuli. Previously, JUNB was shown to act as a critical positive regulator of blood vessel development and homeostasis as well as a negative regulator of proliferation, inflammation and tumour growth. Here, we demonstrate that the oncogenic miR-182 is a novel JUNB target. Loss-of-function studies by morpholino-mediated knockdown and the CRISPR/Cas9 technology identify a novel function for both JUNB and its target miR-182 in lymphatic vascular development in zebrafish. Furthermore, we show that miR-182 attenuates foxo1 expression indicating that strictly balanced Foxo1 levels are required for proper lymphatic vascular development in zebrafish. In conclusion, our findings uncover with the Junb/miR-182/Foxo1 regulatory axis a novel key player in governing lymphatic vascular morphogenesis in zebrafish.

Efficient keratinocyte differentiation strictly depends on JNK-induced soluble factors in fibroblasts.

Previous studies demonstrated that fibroblast-derived and JUN-dependent soluble factors have a crucial role on keratinocyte proliferation and differentiation during cutaneous wound healing. Furthermore, mice with a deficiency in Jun N-terminal kinases (JNKs) , JNK1 or JNK2, showed impaired skin development and delayed wound closure. To decipher the role of dermal JNK in keratinocyte behavior during these processes, we used a heterologous coculture model combining primary human keratinocytes and murine fibroblasts. Although cocultured JNK1/JNK2-deficient fibroblasts did not affect keratinocyte proliferation, temporal monitoring of the transcriptome of differentiating keratinocytes revealed that efficient keratinocyte differentiation not only requires the support by fibroblast-derived soluble factors, but is also critically dependent on JNK1 and JNK2 signaling in these cells. Moreover, we showed that the repertoire of fibroblast transcripts encoding secreted proteins is severely disarranged upon loss of JNK under the coculture conditions applied. Finally, our data demonstrate that efficient keratinocyte terminal differentiation requires constant presence of JNK-dependent and fibroblast-derived soluble factors. Taken together, our results imply that mesenchymal JNK has a pivotal role in the paracrine cross talk between dermal fibroblasts and epidermal keratinocytes during wound healing.

Loss of stromal JUNB does not affect tumor growth and angiogenesis.

The transcription factor AP-1 subunit JUNB has been shown to play a pivotal role in angiogenesis. It positively controls angiogenesis by regulating Vegfa as well as the transcriptional regulator Cbfb and its target Mmp13. In line with these findings, it has been demonstrated that tumor cell-derived JUNB promotes tumor growth and angiogenesis. In contrast to JUNB's function in tumor cells, the role of host-derived stromal JUNB has not been elucidated so far. Here, we show that ablation of Junb in stromal cells including endothelial cells (ECs), vascular smooth muscle cells (SMCs) and fibroblasts does not affect tumor growth in two different syngeneic mouse models, the B16-F1 melanoma and the Lewis lung carcinoma model. In-depth analyses of the tumors revealed that tumor angiogenesis remains unaffected as assessed by measurements of the microvascular density and relative blood volume in the tumor. Furthermore, we could show that the maturation status of the tumor vasculature, analyzed by the SMC marker expression, α-smooth muscle actin and Desmin, as well as the attachment of pericytes to the endothelium, is not changed upon ablation of Junb. Taken together, these results indicate that the pro-angiogenic functions of stromal JUNB are well compensated with regard to tumor angiogenesis and tumor growth.

Ultralarge von Willebrand factor fibers mediate luminal Staphylococcus aureus adhesion to an intact endothelial cell layer under shear stress.

BACKGROUND: During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. METHODS AND RESULTS: Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5). CONCLUSIONS: Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureus-induced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis.

Junb regulates arterial contraction capacity, cellular contractility, and motility via its target Myl9 in mice.

Cellular contractility and, thus, the ability to alter cell shape are prerequisites for a number of important biological processes such as cytokinesis, movement, differentiation, and substrate adherence. The contractile capacity of vascular smooth muscle cells (VSMCs) is pivotal for the regulation of vascular tone and thus blood pressure and flow. Here, we report that conditional ablation of the transcriptional regulator Junb results in impaired arterial contractility in vivo and in vitro. This was exemplified by resistance of Junb-deficient mice to DOCA-salt-induced volume-dependent hypertension as well as by a decreased contractile capacity of isolated arteries. Detailed analyses of Junb-deficient VSMCs, mouse embryonic fibroblasts, and endothelial cells revealed a general failure in stress fiber formation and impaired cellular motility. Concomitantly, we identified myosin regulatory light chain 9 (Myl9), which is critically involved in actomyosin contractility and stress fiber assembly, as a Junb target. Consistent with these findings, reexpression of either Junb or Myl9 in Junb-deficient cells restored stress fiber formation, cellular motility, and contractile capacity. Our data establish a molecular link between the activator protein-1 transcription factor subunit Junb and actomyosin-based cellular motility as well as cellular and vascular contractility by governing Myl9 transcription.

Stromal expression of MMP-13 is required for melanoma invasion and metastasis.

Tumor invasion and metastasis of malignant melanoma have been shown to require proteolytic degradation of the extracellular environment achieved primarily by enzymes of the matrix metalloproteinases (MMP) family. We have earlier shown that increased enzyme activity is localized at the border of tumor cells and the adjacent peritumoral connective tissue, emphasizing the importance of tumor-stroma interactions in the regulation of MMP activity. To confirm the role of stroma-derived MMP-13 in the invasion process, we investigated the invasiveness of melanoma cells upon intradermal injection in mice with complete inactivation of MMP-13. Tumor growth was significantly impaired in mmp-13(-/-) mice and most significant at early time points as compared with wild-type littermates. Moreover, metastasis to various organs was reduced to 17.6 vs 30% in lungs, 2.9 vs 30% in the liver. Strikingly, ablation of MMP-13 completely abrogated formation of metastasis in the heart (0 vs 40%). Notably, decreased tumor growth in mmp-13(-/-) mice was associated with reduced blood vessel density. In addition, decreased blood vessel permeability in the tumors was measured by magnetic resonance imaging of tumor-bearing animals. These data suggest an important role of MMP-13 in tumor growth and an unexpected role in organ-specific metastasis of melanoma cells.

Loss of Drop1 expression already at early tumor stages in a wide range of human carcinomas.

In a study on gene deregulation in ovarian carcinoma we found a mRNA coding for a 350 kDa protein, Drop1, to be downregulated 20- to 180-fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5' region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two-hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors.

JunB is required for IgE-mediated degranulation and cytokine release of mast cells.

Mast cells are effector cells of IgE-mediated immune responses frequently found at the vicinity of blood vessels, the margins of diverse tumors and at sites of potential infection and inflammation. Upon IgE-mediated stimulation, mast cells produce and secrete a broad spectrum of cytokines and other inflammatory mediators. Recent work identified JunB, a member of the AP-1 transcription factor family, as critical regulator of basal and induced expression of inflammatory mediators in fibroblasts and T cells. To study the impact of JunB on mast cell biology, we analyzed JunB-deficient mast cells. Mast cells lacking JunB display a normal in vivo maturation, and JunB-deficient bone marrow cells in vitro differentiated to mast cells show no alterations in proliferation or apoptosis. But these cells exhibit impaired IgE-mediated degranulation most likely due to diminished expression of SWAP-70, Synaptotagmin-1, and VAMP-8, and due to impaired influx of extracellular calcium. Moreover, JunB-deficient bone marrow mast cells display an altered cytokine expression profile in response to IgE stimulation. In line with these findings, the contribution of JunB-deficient mast cells to angiogenesis, as analyzed in an in vitro tube formation assay on matrigel, is severely impaired due to limiting amounts of synthesized and secreted vascular endothelial growth factor. Thus, JunB is a critical regulator of intrinsic mast cell functions including cross-talk with endothelial cells.

Infarct volume after transient middle cerebral artery occlusion (MCAo) can be reduced by attenuation but not by inactivation of c-Jun action.

Stroke therapy aims to save penumbral tissue from apoptosis that is activated in response to the ischemic injury. Since the c-Jun transcription factor plays a crucial role in promoting apoptosis, inhibition of its activation might reduce the final infarct size and thus increase functional outcome. To test this hypothesis we made use of four genetically modified mouse lines influencing the c-Jun pathway at various steps. Upon transient middle cerebral artery occlusion for 90 min and 24 h of reperfusion, infarct volume and number of ATF-2-, TUNEL- and cleaved Caspase-3-positive cells were determined in conditional c-Jun knock-out mice (cond. c-Jun), mice overexpressing JunB (JunBtg), mice lacking the phosphoacceptor serines 63 and 73 of c-Jun (JunAA) and in mice overexpressing Bcl-2 (Bcl-2tg). Cond. c-Jun as well as JunAA mice did not show significant differences in the infarct size when compared to their non-mutant controls. By contrast smaller infarct volumes were detected in transgenic mice merely attenuating c-Jun action (JunBtg and Bcl-2tg). ATF-2, TUNEL or cleaved Caspase-3 staining revealed no significant differences between the experimental groups. A complete lack of functional c-Jun might be compensated by other cellular mechanisms, in contrast to its reduced function. Thus, our data suggest that attenuation rather than a complete block of c-Jun action appears to be more promising for therapy of stroke.

JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells.

Heme oxygenase-1 is a highly inducible gene, the product of which catalyzes breakdown of the prooxidant heme. The purpose of this study was to investigate the regulation of the human heme oxygenase-1 gene in renal epithelial cells. DNase I hyper-sensitivity studies identified three distal sites (HS-2, -3, and -4) corresponding to approximately -4.0, -7.2, and -9.2 kb, respectively, of the heme oxygenase-1 promoter in addition to one proximal region, HS-1, which we have shown previously to be an E box. In vivo dimethyl sulfate footprinting of the HS-2 region revealed six individual protected guanines. Two mutations within HS-2 combined with a third mutation of the proximal E box abolished hemin- and cadmium-driven heme oxygenase-1 promoter activation, suggesting that these three sites synergized for maximal heme oxygenase-1 induction. Jun proteins bound to the antioxidant response element in the HS-2 region in vitro and associated with the heme oxygenase-1 promoter in vivo. JunB and JunD contribute opposing effects; JunB activated whereas JunD repressed heme oxygenase-1 expression in human renal epithelial cells, results that were corroborated in junB(-)(/)(-) and junD(-)(/)(-) cells. We propose that heme oxygenase-1 induction is controlled by a dynamic interplay of regulatory proteins, and we provide new insights into the molecular control of the human heme oxygenase-1 gene.

Critical role for NF-kappaB-induced JunB in VEGF regulation and tumor angiogenesis.

Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP-1 transcription factor is considered as facilitator of hypoxia-induced VEGF expression through interaction with hypoxia-inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP-1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP-1 subunit junB is a target gene of hypoxia-induced signaling via NF-kappaB. Loss of JunB in various cell types results in severely impaired hypoxia-induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB-deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB(-/-) teratocarcinomas because of severely impaired paracrine-acting VEGF and the subsequent inability to efficiently recruit host-derived vessels.

JunB is required for endothelial cell morphogenesis by regulating core-binding factor beta.

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor beta (CBFbeta), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFbeta into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFbeta in EC morphogenesis.

Delayed wound healing and epidermal hyperproliferation in mice lacking JunB in the skin.

The cutaneous response to injury and stress comprises a temporary change in the balance between epidermal proliferation and differentiation as well as an activation of the immune system. Soluble factors play an important role in the regulation of these complex processes by coordinating the intercellular communication between keratinocytes, fibroblasts, and inflammatory cells. In this study, we demonstrate that JunB, a member of the activator protein-1 transcription factor family, is an important regulator of cytokine expression and thus critically involved in the cutaneous response to injury and stress. Mice lacking JunB in the skin develop normally, indicating that JunB is neither required for cutaneous organogenesis, nor homeostasis. However, upon wounding and treatment with the phorbol ester 12-O-decanoyl-phorbol-13-acetate, JunB-deficiency in the skin likewise resulted in pronounced epidermal hyperproliferation, disturbed differentiation, and prolonged inflammation. Furthermore, delayed tissue remodelling was observed during wound healing. These phenotypic skin abnormalities were associated with JunB-dependent alterations in expression levels and kinetics of important mediators of wound repair, such as granulocyte macrophage colony-stimulating factor, growth-regulated protein-1, macrophage inflammatory protein-2, and lipocalin-2 in both the dermal and epidermal compartment of the skin, and a reduced ability of wound contraction of mutant dermal fibroblasts in vitro.

Cutting edge: the AP-1 subunit JunB determines NK cell-mediated target cell killing by regulation of the NKG2D-ligand RAE-1epsilon.

The activating receptor NKG2D and its ligands RAE-1 play an important role in the NK, gammadelta+, and CD8+ T cell-mediated immune response to tumors. Expression levels of RAE-1 on target cells have to be tightly controlled to allow immune cell activation against tumors but to avoid destruction of healthy tissues. In this study, we report that cell surface expression of RAE-1epsilon is greatly enhanced on cells lacking JunB, a subunit of the transcription complex AP-1. Furthermore, tissue-specific junB knockout mice respond to 12-O-tetradecanoyl-phorbol-13-acetate, a potent AP-1 activator, with markedly increased and sustained epidermal RAE-1epsilon expression. Accordingly, junB-deficient cells are efficiently killed via NKG2D by NK cells and induce IFN-gamma production. Our data indicate that the transcription factor AP-1, which is involved in tumorigenesis and cellular stress responses, regulates RAE-1epsilon. Thus, up-regulated RAE-1epsilon expression due to low levels of JunB could alert immune cells to tumors and stressed cells.

Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice.

Degradation of the extracellular matrix, which is an indispensable step in tissue remodelling processes such as embryonic development and wound healing of the skin, has been attributed to collagenolytic activity of members of the matrix metalloproteinase family (MMPs). Here, we employed mmp13 knockout mice to elucidate the function of MMP13 in embryonic skin development, skin homeostasis, and cutaneous wound healing. Overall epidermal architecture and dermal composition of non-injured skin were indistinguishable from wild-type mice. Despite robust expression of MMP13 in the early phase of wound healing, wild-type and mmp13 knockout animals did not differ in their efficiency of re-epithelialization, inflammatory response, granulation tissue formation, angiogenesis, and restoration of basement membrane. Yet, among other MMPs also expressed during wound healing, MMP8 was found to be enhanced in wounds of MMP13-deficient mice. In summary, skin homeostasis and also tissue remodelling processes like embryonic skin development and cutaneous wound healing are independent of MMP13 either owing to MMP13 dispensability or owing to functional substitution by other collagenolytic proteinases such as MMP8.