The hidden hedgehog of the pituitary: hedgehog signaling in development, adulthood and disease of the hypothalamic-pituitary axis.

Hedgehog signaling plays pivotal roles in embryonic development, adult homeostasis and tumorigenesis. However, its engagement in the pituitary gland has been long underestimated although Hedgehog signaling and pituitary embryogenic development are closely linked. Thus, deregulation of this signaling pathway during pituitary development results in malformation of the gland. Research of the last years further implicates a regulatory role of Hedgehog signaling in the function of the adult pituitary, because its activity is also interlinked with homeostasis, hormone production, and most likely also formation of neoplasms of the gland. The fact that this pathway can be efficiently targeted by validated therapeutic strategies makes it a promising candidate for treating pituitary diseases. We here summarize the current knowledge about the importance of Hedgehog signaling during pituitary development and review recent data that highlight the impact of Hedgehog signaling in the healthy and the diseased adult pituitary gland.

Tumor suppressive functions of WNT5A in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly aggressive soft tissue malignancy that predominantly affects children. The main subtypes are alveolar RMS (ARMS) and embryonal RMS (ERMS) and the two show an impaired muscle differentiation phenotype. One pathway involved in muscle differentiation is WNT signaling. However, the role of this pathway in RMS is far from clear. Our recent data showed that the canonical WNT/ß­Catenin pathway serves a subordinate role in RMS, whereas non­canonical WNT signaling probably is more important for this tumor entity. The present study investigated the role of WNT5A, which is the major ligand of non­canonical WNT signaling, in ERMS and ARMS. Gene expression analysis showed that WNT5A was expressed in human RMS samples and that its expression is more pronounced in ERMS. When stably overexpressed in RMS cell lines, WNT5A decreased proliferation and migration of the cells as demonstrated by BrdU incorporation and Transwell migration or scratch assay, respectively. WNT5A also decreased the self­renewal capacity and the expression of stem cell markers and modulates the levels of muscle differentiation markers as shown by sphere assay and western blot analysis, respectively. Finally, overexpression of WNT5A can destabilize active ß­Catenin of RMS cells. A WNT5A knockdown has opposite effects. Together, the results suggest that WNT5A has tumor suppressive functions in RMS, which accompanies downregulation of ß­Catenin.

Oncogenic NRAS Accelerates Rhabdomyosarcoma Formation When Occurring within a Specific Time Frame during Tumor Development in Mice.

In the Ptch+/- mouse model for embryonal rhabdomyosarcoma (ERMS), we recently showed that oncogenic (onc) H-, K- or NRAS mutations do not influence tumor growth when induced at the advanced, full-blown tumor stage. However, when induced at the invisible ERMS precursor stage at 4 weeks of age, tumor development was enforced upon oncHRAS and oncKRAS but not by oncNRAS, which instead initiated tumor differentiation. These data indicate that oncRAS-associated processes differ from each other in dependency on the isoform and their occurrence during tumor development. Here, we investigated the outcome of oncNRAS induction at an earlier ERMS precursor stage at 2 weeks of age. In this setting, oncNRAS accelerates tumor growth because it significantly shortens the ERMS-free survival and increases the ERMS incidence. However, it does not seem to alter the differentiation of the tumors. It is also not involved in tumor initiation. Together, these data show that oncNRAS mutations can accelerate tumor growth when targeting immature ERMS precursors within a specific time window, in which the precursors are permissive to the mutation and show that oncNRAS-associated processes differ from each other in dependency on their occurrence during tumor development.

Context-dependent modulation of aggressiveness of pediatric tumors by individual oncogenic RAS isoforms.

A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS, KRAS, or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh-driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse.

Hedgehog signaling in endocrine and folliculo-stellate cells of the adult pituitary.

Ubiquitous overactivation of Hedgehog signaling in adult pituitaries results in increased expression of proopiomelanocortin (Pomc), growth hormone (Gh) and prolactin (Prl), elevated adrenocorticotropic hormone (Acth) production and proliferation of Sox2+ cells. Moreover, ACTH, GH and PRL-expressing human pituitary adenomas strongly express the Hedgehog target GLI1. Accordingly, Hedgehog signaling seems to play an important role in pathology and probably also in homeostasis of the adult hypophysis. However, the specific Hedgehog-responsive pituitary cell type has not yet been identified. We here investigated the Hedgehog pathway activation status and the effects of deregulated Hedgehog signaling cell-specifically in endocrine and non-endocrine pituitary cells. We demonstrate that Hedgehog signaling is unimportant for the homeostasis of corticotrophs, whereas it is active in subpopulations of somatotrophs and folliculo-stellate cells in vivo. Reinforcement of Hedgehog signaling activity in folliculo-stellate cells stimulates growth hormone production/release from somatotrophs in a paracrine manner, which most likely is mediated by the neuropeptide vasoactive intestinal peptide. Overall, our data show that Hedgehog signaling affects the homeostasis of pituitary hormone production via folliculo-stellate cell-mediated regulation of growth hormone production/secretion.

Heterozygous truncating variants in SUFU cause congenital ocular motor apraxia.

PURPOSE: This study aimed to delineate the genetic basis of congenital ocular motor apraxia (COMA) in patients not otherwise classifiable. METHODS: We compiled clinical and neuroimaging data of individuals from six unrelated families with distinct clinical features of COMA who do not share common diagnostic characteristics of Joubert syndrome or other known genetic conditions associated with COMA. We used exome sequencing to identify pathogenic variants and functional studies in patient-derived fibroblasts. RESULTS: In 15 individuals, we detected familial as well as de novo heterozygous truncating causative variants in the Suppressor of Fused (SUFU) gene, a negative regulator of the Hedgehog (HH) signaling pathway. Functional studies showed no differences in cilia occurrence, morphology, or localization of ciliary proteins, such as smoothened. However, analysis of expression of HH signaling target genes detected a significant increase in the general signaling activity in COMA patient-derived fibroblasts compared with control cells. We observed higher basal HH signaling activity resulting in increased basal expression levels of GLI1, GLI2, GLI3, and Patched1. Neuroimaging revealed subtle cerebellar changes, but no full-blown molar tooth sign. CONCLUSION: Taken together, our data imply that the clinical phenotype associated with heterozygous truncating germline variants in SUFU is a forme fruste of Joubert syndrome.

Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice.

Basal cell carcinoma (BCC) originate from Hedgehog/Patched signaling-activated epidermal stem cells. However, the chemically induced tumorigenesis of mice with a CD4Cre-mediated biallelic loss of the Hedgehog signaling repressor Patched also induces BCC formation. Here, we identified the cellular origin of CD4Cre-targeted BCC progenitors as rare Keratin 5+ epidermal cells and show that wildtype Patched offspring of these cells spread over the hair follicle/skin complex with increasing mouse age. Intriguingly, Patched mutant counterparts are undetectable in age-matched untreated skin but are getting traceable upon applying the chemical tumorigenesis protocol. Together, our data show that biallelic Patched depletion in rare Keratin 5+ epidermal cells is not sufficient to drive BCC development, because the spread of these cells is physiologically suppressed. However, bypassing the repression of Patched mutant cells, e.g., by exogenous stimuli, leads to an accumulation of BCC precursor cells and, finally, to tumor development.

WIF1 Suppresses the Generation of Suprabasal Cells in Acanthotic Skin and Growth of Basal Cell Carcinomas upon Forced Overexpression.

We analyzed the role of WIF1 in normal and acanthotic epidermis of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans-retinoic acid (ATRA)-treated and basal cell carcinoma (BCC)-bearing mice. WIF1 protein is located in the follicular infundibulum and interfollicular epidermis (IFE) in murine back skin. Within the hyperplastic epidermis of TPA- or ATRA-treated or BCC-bearing murine skin, WIF1 and Keratin 10 overlap in Ki67⁻ suprabasal layers, while basal epidermal layers expressing Ki67, and BCCs expressing Wif1 mRNA, are free of WIF1 protein. This is similar in human skin, with the exception that WIF1 protein is found in single Ki67⁻ basal epidermal cells in normal skin and additionally in Ki67+ cells in acanthotic skin. Wif1-deficiency enhances acanthosis of the murine BCC-associated epidermis, which is accompanied by an increase of Ki67+ and of Sca-1+ basal cells. WIF1 overexpression in allografted BCC-derived keratinocytes prevents growth and keratinization, involving enhanced phosphorylation of protein kinase C and extracellular signal-regulated kinase 1 and arguably factors secreted by the in vivo environment. In summary, WIF1 protein marks suprabasal layers in the normal IFE. It is also present in the epidermis overlaying BCCs where it diminishes proliferation of basal cells and production of differentiating suprabasal cells. In addition, WIF1 can prevent proliferation and keratinization of BCC-related keratinocytes.

Regulation and Role of GLI1 in Cutaneous Squamous Cell Carcinoma Pathogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin tumor in humans. Although current therapies are sufficient to clear the tumor in many cases, the overall risk of cSCC metastasis is still 5%. Alternative treatment options could help to overcome this situation. Here we focused on the role of the Hedgehog (HH) signaling pathway and its interplay with epidermal growth factor receptor (EGFR) signaling in cSCC. The analyses revealed that, despite lack of Sonic HH (SHH) expression, a subset of human cSCC can express GLI1, a marker for active HH signaling, within distinct tumor areas. In contrast, all tumors strongly express EGFR and the hair follicle stem cell marker SOX9 at the highly proliferative tumor-stroma interface, whereas central tumor regions with a more differentiated stratum spinosum cell type lack both EGFR and SOX9 expression. In vitro experiments indicate that activation of EGFR signaling in the human cSCC cell lines SCL-1, MET-1, and MET-4 leads to GLI1 inhibition via the MEK/ERK axis without affecting cellular proliferation. Of note, EGFR activation also inhibits cellular migration of SCL-1 and MET-4 cells. Because proliferation and migration of the cells is also not altered by a GLI1 knockdown, GLI1 is apparently not involved in processes of aggressiveness in established cSCC tumors. In contrast, our data rather suggest a negative correlation between Gli1 expression level and cSCC formation because skin of Ptch +/- mice with slightly elevated Gli1 expression levels is significantly less susceptible to chemically-induced cSCC formation compared to murine wildtype skin. Although not yet formally validated, these data open the possibility that GLI1 (and thus HH signaling) may antagonize cSCC initiation and is not involved in cSCC aggressiveness, at least in a subset of cSCC.

Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model.

Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12⁻15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability.

Different Response of Ptch Mutant and Ptch Wildtype Rhabdomyosarcoma Toward SMO and PI3K Inhibitors.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.

Indian Hedgehog Suppresses a Stromal Cell-Driven Intestinal Immune Response.

BACKGROUND & AIMS: Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation. METHODS: Hedgehog activity was modulated genetically in both cell type-specific and body-wide models and the resulting animals were analyzed for gene expression profiles and sensitivity for dextran sodium sulfate (DSS) colitis. To characterize the Hedgehog target cell, Gli1-CreERT2-Rosa26-ZsGreen animals were generated, which express ZsGreen in all Hedgehog-responsive cells. These cells were characterized using flow cytometry and immunofluorescence. RESULTS: Loss of Indian Hedgehog from the intestinal epithelium resulted in a rapid increase in expression of inflammation-related genes, accompanied by increased influx of immune cells. Animals with epithelium-specific deletion of Ihh or lacking the Hedgehog receptor Smoothened from Hedgehog target cells were more sensitive to DSS colitis. In contrast, specific deletion of Smoothened in the myeloid compartment did not alter the response to DSS. This suggests that Hedgehog signaling does not repress intestinal immunity through an effect on myeloid cells. Indeed, we found that Hedgehog-responsive cells expressed gp38, smooth muscle actin, and desmin, indicating a fibroblastic nature. Ihh signaling inhibited expression of C-X-C motif chemokine ligand 12 (CXCL12) in fibroblasts in vitro and in vivo, thereby impairing the recruitment of immune cells. CONCLUSIONS: We show that epithelium-derived Indian Hedgehog signals exclusively to fibroblasts in the intestine. Loss of Ihh leads to a rapid immune response with up-regulation of fibroblast-derived CXCL12, and migration of immune cells into the lamina propria.

Hedgehog signaling activation induces stem cell proliferation and hormone release in the adult pituitary gland.

Hedgehog (HH) signaling is known to be essential during the embryonal development of the pituitary gland but the knowledge about its role in the adult pituitary and in associated tumors is sparse. In this report we investigated the effect of excess Hh signaling activation in murine pituitary explants and analyzed the HH signaling status of human adenopituitary lobes and a large cohort of pituitary adenomas. Our data show that excess Hh signaling led to increased proliferation of Sox2(+) and Sox9(+) adult pituitary stem cells and to elevated expression levels of adrenocorticotropic hormone (Acth), growth hormone (Gh) and prolactin (Prl) in the adult gland. Inhibition of the pathway by cyclopamine reversed these effects indicating that active Hh signaling positively regulates proliferative processes of adult pituitary stem cells and hormone production in the anterior pituitary. Since hormone producing cells of the adenohypophysis as well as ACTH-, GH- and PRL-immunopositive adenomas express SHH and its target GLI1, we furthermore propose that excess HH signaling is involved in the development/maintenance of hormone-producing pituitary adenomas. These findings advance the understanding of physiological hormone regulation and may open new treatment options for pituitary tumors.

A Functional and Putative Physiological Role of Calcitriol in Patched1/Smoothened Interaction.

The Patched1 (Ptch)-mediated inhibition of Smoothened (Smo) is still an open question. However, a direct Ptch/Smo interaction has been excluded, Smo modulators were identified, but the endogenous signal transmitting molecule remains undiscovered. Here, we demonstrate that calcitriol, the hormonally active form of vitamin D3, is an excellent candidate for transmission of Ptch/Smo interaction. Our study reveals that Ptch expression is sufficient to release calcitriol from the cell and that calcitriol inhibits Smo action and ciliary translocation by acting on a site distinct from the 7-transmembrane domain or the cysteine-rich domain. Moreover calcitriol strongly synergizes with itraconazole (ITZ) in Smo inhibition, which did not result from elevated calcitriol bioavailability due to ITZ-mediated 24-hydroxylase inhibition but rather from a direct interaction of the compounds at the level of Smo. Together, we suggest that calcitriol represents a possible endogenous transmitter of Ptch/Smo interaction. Moreover calcitriol or calcitriol derivatives combined with ITZ might be a treatment option of Hedgehog-associated cancers.

Depletion of cutaneous macrophages and dendritic cells promotes growth of basal cell carcinoma in mice.

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.

Inactivation of patched1 in murine chondrocytes causes spinal fusion without inflammation.

OBJECTIVE: During development of the vertebrate skeleton, chondrocytes form a cartilage template that is gradually replaced by bone. Hormones of the Hedgehog (HH) family have been implicated in the ossification process, but their exact relationship to normal or pathogenic bone formation is unclear. This study was undertaken to establish a genetic tool that allows the discrete inactivation of genes in spinal chondrocytes, and to investigate in vivo how chondrocyte-specific ablation of the inhibitory HH receptor Patched 1 (Ptch1) affects skeleton integrity. METHODS: A Cre-deleter mouse strain, mb1-Cre, for selective gene recombination in spinal chondrocytes was identified by in situ hybridization and histologic analysis. The mb1-Cre(+/-) animals were crossed with mice that harbor a loxP-flanked Ptch1 gene (Ptch1(flox/flox) ) to abrogate the inhibition of the HH signaling pathway in chondrocytes. The skeletal integrity of F1 mice was characterized by high-resolution flat-panel-based volume computed tomography and histologic staining procedures. RESULTS: During the first weeks after birth, all mb1-Cre(+/-) /Ptch1(flox/flox) mice developed progressive spinal fusion with malformation of the vertebrae. This phenotype was caused by aberrant chondrocyte proliferation in the intervertebral discs that blocked endochondral ossification. Importantly, the disease pattern occurred in an inflammation-independent manner. CONCLUSION: Our findings indicate that chronic activation of the HH signal pathway in spinal chondrocytes can trigger an ankylosing spine morphology without immune cell contributions. Hence, the destruction of cartilage and loss of axial joint integrity can result from chondrocyte-intrinsic defects of monogenic origin.

DMBA/TPA treatment is necessary for BCC formation from patched deficient epidermal cells in Ptch(flox/flox)CD4Cre(+/-) mice.

The development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptch(flox/flox)CD4Cre(+/-) mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptch(flox/flox)CD4Cre(+/-) mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4(+) cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptch(flox/flox)CD4Cre(+/-) BCC mouse model.

The hedgehog receptor patched1 in T cells is dispensable for adaptive immunity in mice.

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and Treg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo.

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.

Inactivation of Patched1 in mice leads to development of gastrointestinal stromal-like tumors that express Pdgfrα but not kit.

BACKGROUND & AIMS: A fraction of gastrointestinal stromal tumor (GIST) cells overexpress the platelet-derived growth factor receptor (PDGFR)A, although most overexpress KIT. It is not known if this is because these receptor tyrosine kinases have complementary oncogenic potential, or because of heterogeneity in the cellular origin of GIST. Little also is known about why Hedgehog (HH) signaling is activated in some GIST. HH binds to and inactivates the receptor protein patched homolog (PTCH). METHODS: Ptch was conditionally inactivated in mice (to achieve constitutive HH signaling) using a Cre recombinase regulated by the lysozyme M promoter. Cre-expressing cells were traced using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, immunoblot, and quantitative reverse-transcriptase polymerase chain reaction analyses. Cell transformation was assessed by soft agar assay. RESULTS: Loss of Ptch from lysozyme M-expressing cells resulted in the development of tumors of GIST-like localization and histology; these were reduced when mice were given imatinib, a drug that targets KIT and PDGFRA. The Hh signaling pathway was activated in the tumor cells, and Pdgfrα, but not Kit, was overexpressed and activated. Lineage tracing revealed that Cre-expressing intestinal cells were Kit-negative. These cells sometimes expressed Pdgfrα and were located near Kit-positive interstitial cells of Cajal. In contrast to KIT, activation of PDGFRA increased anchorage-independent proliferation and was required for tumor formation in mice by cells with activated HH signaling. CONCLUSIONS: Inactivation of Ptch in mice leads to formation of GIST-like tumors that express Pdgfrα, but not Kit. Activation of Pdgfrα signaling appears to facilitate tumorigenesis.