Tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice.

Psoriasis is a common chronic inflammatory skin disease with a prevalence of about 2% in the Caucasian population. Tumor necrosis factor (TNF) plays an essential role in the pathogenesis of psoriasis, but its mechanism of action remains poorly understood. Here we report that the development of psoriasis-like skin inflammation in mice with epidermis-specific inhibition of the transcription factor NF-κB was triggered by TNF receptor 1 (TNFR1)-dependent upregulation of interleukin-24 (IL-24) and activation of signal transducer and activator of transcription 3 (STAT3) signaling in keratinocytes. IL-24 was strongly expressed in human psoriatic epidermis, and pharmacological inhibition of NF-κB increased IL-24 expression in TNF-stimulated human primary keratinocytes, suggesting that this mechanism is relevant for human psoriasis. Therefore, our results expand current views on psoriasis pathogenesis by revealing a new keratinocyte-intrinsic mechanism that links TNFR1, NF-κB, ERK, IL-24, IL-22R1, and STAT3 signaling to disease initiation.

Severe liver disease resembling PSC in mice with K5-Cre mediated deletion of Krüppel-like factor 5 (Klf5).

Chronic cholestatic liver diseases including primary sclerosing cholangitis (PSC) present a complex spectrum with regards to the cause, age of manifestation and histopathological features. Current treatment options are severely limited primarily due to a paucity of model systems mirroring the disease. Here, we describe the Keratin 5 (K5)-Cre; Klf5fl/fl mouse that spontaneously develops severe liver disease during the postnatal period with features resembling PSC including a prominent ductular reaction, fibrotic obliteration of the bile ducts and secondary degeneration/necrosis of liver parenchyma. Over time, there is an expansion of Sox9+ hepatocytes in the damaged livers suggestive of a hepatocyte-mediated regenerative response. We conclude that Klf5 is required for the normal function of the hepatobiliary system and that the K5-Cre; Klf5fl/fl mouse is an excellent model to probe the molecular events interlinking damage and regenerative response in the liver.

GLI1-induced mammary gland tumours are transplantable and maintain major molecular features.

Increased expression of GLI1, the main Hedgehog signalling pathway effector, is related to unfavourable prognosis and progressive disease of certain breast cancer subtypes. We used conditional transgenic mice induced to overexpress GLI1 in the mammary epithelium either alone or in combination with deletion of one Trp53 allele to address the role of elevated GLI1 expression in breast tumour initiation and progression. Induced GLI1 expression facilitates mammary gland tumour formation and this was further increased upon heterozygous deletion of Trp53. The GLI1-induced primary tumours were of different murine molecular subtypes, including Normal-likeEx , Class8Ex , Claudin-LowEx and Erbb2-likeEx . The gene expression profiles of some of the tumours correlated well with the PAM50 subtypes for human breast cancer. Whole-exome sequencing revealed somatic mutation profiles with only little overlap between the primary tumours. Orthotopically serially transplanted GLI1-induced tumours maintained the main morphological characteristics of the primary tumours for ≥10 generations. Independent of Trp53 status and molecular subtype, the serially transplanted GLI1-induced tumours were able to grow both in the absence of transgenic GLI1 expression and in the presence of the GLI1 inhibitor GANT61. These data suggest that elevated GLI1 expression has a determinant role in tumour initiation; however, additional genetic events are required for tumour progression.

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development.

Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.

Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model.

Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.

A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

ß-Catenin-Gli1 interaction regulates proliferation and tumor growth in medulloblastoma.

BACKGROUND: The Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB). In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known. FINDINGS: Here we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice (Ptch(+/-)) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase (SA-betaGal). Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as "tumor take". Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining. CONCLUSION: We propose that beta-catenin stabilization increases its physical interaction with Gli1, leading to Gli1 degradation and inhibition of Hh signaling, thereby promoting tumor cell senescence and suppression of "tumor take" in mice.

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus.

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Wounding enhances epidermal tumorigenesis by recruiting hair follicle keratinocytes.

Chronic wounds and acute trauma constitute well-established risk factors for development of epithelial-derived skin tumors, although the underlying mechanisms are largely unknown. Basal cell carcinomas (BCCs) are the most common skin cancers displaying a number of features reminiscent of hair follicle (HF)-derived cells and are dependent on deregulated Hedgehog (Hh)/GLI signaling. Here we show, in a mouse model conditionally expressing GLI1 and in a model with homozygous inactivation of Ptch1, mimicking the situation in human BCCs, that the wound environment accelerates the initiation frequency and growth of BCC-like lesions. Lineage tracing reveals that both oncogene activation and wounding induce emigration of keratinocytes residing in the lower bulge and the nonpermanent part of the HFs toward the interfollicular epidermis (IFE). However, only oncogene activation in combination with a wound environment enables the participation of such cells in the initiation of BCC-like lesions at the HF openings and in the IFE. We conclude that, in addition to the direct enhancement of BCC growth, the tumor-promoting effect of the wound environment is due to recruitment of tumor-initiating cells originating from the neighboring HFs, establishing a link between epidermal wounds and skin cancer risk.

Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus.

OBJECTIVE: In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. DESIGN: The authors used transgenic mice in which the Hh receptor Patched1 (Ptch1) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. RESULTS: Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. CONCLUSION: Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. METHODS: Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. RESULTS: Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. CONCLUSIONS: We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.

Loss of Trp53 promotes medulloblastoma development but not skin tumorigenesis in Sufu heterozygous mutant mice.

Basal cell carcinoma of the skin typically carries genetic alterations in components of the hedgehog (HH) signaling pathway. Previously, we generated a knockout mouse with a loss-of-function mutation in suppressor of fused (Sufu), an essential repressor of the pathway downstream of Hh ligand cell surface reception. Mice heterozygous for the mutated Sufu allele develop a skin phenotype that includes lesions similar to basaloid follicular hamartomas. The purpose of the current study was to test the possibility that the simultaneous loss of the tumor suppressor gene, transformation related protein 53 (Trp53), would aggravate the Sufu skin phenotype since Trp53 loss is known to enhance the growth of other Hh-driven tumors. Consistent with previous reports, medulloblastomas and rhabdomyosarcomas developed in Sufu(+/-) ;Trp53(-/-) mice. However, the characteristic Sufu(+/-) skin phenotype was not altered in the absence of Trp53, and showed no changes in latency, multiplicity, cellular phenotype, or proliferative capacity of the basaloid lesions. This finding was both novel and intriguing and demonstrated a differential, tissue-specific sensitivity to Sufu and Trp53 tumor suppressor gene loss, which may be linked to developmental stage and the degree of proliferative activity in specific cell types.

Targeted expression of GLI1 in the salivary glands results in an altered differentiation program and hyperplasia.

Hedgehog (Hh) signaling is a regulator of salivary gland morphogenesis, but its role in postnatal glands has only recently begun to be addressed. To examine the effects of deregulated Hh signaling in the salivary gland, we expressed the Hh effector protein GLI1, in salivary epithelial cells using both cytokeratin 5 and mouse mammary tumor virus (MMTV) transgenic systems. Ectopic pathway activation resulted in restrained acinar differentiation, formation of cystic lesions, and prominent appearance of ductal structures. Moreover, induced expression of GLI1 aids the formation of hyperplastic lesions, which closely resemble GLI1-induced changes in murine skin and mammary glands, suggesting that GLI1 targets cells with similar characteristics in different tissues. Furthermore, GLI1-expressing salivary epithelial cells are actively dividing, and GLI1-induced lesions are proliferative, an incident accompanied by enhanced expression of the Hh target genes, cyclin D1, and Snail. GLI1-induced salivary lesions regress after transgene withdrawal and become histologically normalized. Taken together, our data reveal the ability of GLI1 to modulate salivary acinar differentiation and to promote proliferation of ductal epithelial cells.

Hedgehog beyond medulloblastoma and basal cell carcinoma.

The Hedgehog (Hh) signaling pathway is of central importance during embryo development in metazoans and governs a diverse array of processes including cell proliferation, differentiation, and tissue patterning. In normal adult physiology, the pathway is implicated in stem cell maintenance, tissue repair and regeneration. However, the pathway's darker side is its involvement in several types of human cancer, to which it confers growth promoting and/or survival capabilities to the cancer cell to varying degrees, and by different mechanisms. The Hh pathway is firmly linked to the etiology of basal cell carcinoma and to at least a subset of medulloblastoma. There is increasing evidence that other sporadic cancers, including those in pancreas, prostate, lung, and breast, could also be dependent on Hh pathway activity. In this review, we provide an overview of the pathway's role in various tumor types, where much of the framework for Hh-dependent malignancies has been elucidated in experimental mouse models. We discuss three different signal transduction models for the pathway's involvement in cancer: i) ligand-independent signaling, ii) ligand-dependent autocrine/juxtacrine signaling, and iii) ligand-dependent paracrine signaling. These different modes of signaling may have implications for future therapeutic interventions aimed at inhibiting the pathway during disease. In addition, crosstalk with other pathways, and indications of non-canonical Hh signaling in cancer cells may further cause complications, or perhaps possibilities, in the treatment regimen. Finally, we review the rapid progress and promising results in the development of small-molecule inhibitors of the Hh pathway.

Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway.

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at approximately E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu(-/-) embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu(-/-) fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu(+/-) mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.

The hair follicle-a stem cell zoo.

Recent studies on stem cells in the adult hair follicle (HF) have uncovered a veritable menagerie of exceptionally diverse and dynamic keratinocytes with stem cell properties located in distinct regions of the HF. Although endowed with specific functions during normal hair follicle maintenance, the majority of these cells can act as multipotent stem cells in stress situations, such as physical injury, which argues for an unanticipated degree of plasticity of these cells. This review provides an overview of the different epithelial stem cell populations, identified in the mouse HF, and their relationships with one another, and envisions possible cellular mechanisms underlying normal HF maintenance and skin regeneration.

Antipsychotic drugs regulate hedgehog signaling by modulation of 7-dehydrocholesterol reductase levels.

Recently we identified GANT61, a small-molecule antagonist of Gli transcription factors, which are the final effectors of the mammalian Hedgehog (HH) signaling pathway. Here we describe a diamine substructure of GANT61 that carries the biological activity and show that this part of the molecule is structurally related to trans-1,4-bis(2-chlorobenzaminomethyl)cyclohexane dihydrochloride (AY9944), an inhibitor of the enzymatic activity and transcriptional inducer of 7-dehydrocholesterol-reductase (Dhcr7, EC 1.3.1.21). Treatment of cells with the GANT61 diamine, AY9944, or overexpression of DHCR7 results in the attenuation of Smoothened-dependent and -independent HH signaling. Whereas GANT61 function is independent of Dhcr7, AY9944 does require up-regulation of endogenous Dhcr7. In line with these findings, Dhcr7-modulating antipsychotic (clozapine, chlorpromazine, haloperidol) and antidepressant (imipramine) drugs regulate HH signaling in vitro and in vivo. Modulation of HH signaling may represent a hitherto undiscovered biological (side) effect of therapeutics used to treat schizophrenia and depression.

Suppressor of Fused inhibits mammalian Hedgehog signaling in the absence of cilia.

The Hedgehog (Hh) family of secreted proteins regulates mammalian development and cancer formation through Gli transcription factors, which exist in both activator and repressor forms. In vertebrates, the primary cilia play an essential role in Hh signal transduction and are required for both the activator and repressor activities of Gli proteins. In the current study, we demonstrate that mouse Suppressor of Fused (Sufu) interacts with Gli proteins and inhibits Gli activator activity in the absence of cilia. Removal of Sufu in both Smoothened (Smo) and Ift88 mutants, respectively, leads to full activation of Hh signaling, suggesting that Smo-mediated repression of Sufu, but not the inhibitory function of Sufu, requires cilia. Finally, we show that Sufu is important for proper activator/repressor ratio of Gli3 protein in mice, both in the presence and absence of cilia.