Dynamics of notch expression during murine prostate development and tumorigenesis.

Notch signaling has been widely demonstrated to be responsible for cell fate determination during normal development and implicated in human T-cell leukemia and mouse mammary carcinomas. Here we show that Notch signaling may be involved in prostatic development and cancer cell growth. In situ hybridization and reverse transcription-PCR analyses revealed that Notch1 was expressed in prostate epithelial cells during normal development and in prostate cancer cells. Characterization of Notch1-green fluorescent protein transgenic mice, in which the expression of reporter green fluorescent protein is under the control of the Notch1 promoter, indicated that Notch1-expressing cells were associated with the basal epithelial cell population in the prostate. Examination of the transgenic adenocarcinoma of the mouse prostate showed that expression of Notch1 was elevated in malignant prostatic epithelial cells of primary and metastatic tumors. Expression of Notch ligands, however, was low or undetectable in cultured prostate cancer cells or in malignant prostatic epithelial cells in transgenic adenocarcinoma of the mouse prostate. Furthermore, overexpression of a constitutively active form of Notch1 inhibited the proliferation of various prostate cancer cells, including DU145, LNCaP, and PC3 cells. Taken together, our data indicate for the first time that Notch signaling may play a role in murine prostatic development and tumorigenesis.

Multiple effects of artemin on sympathetic neurone generation, survival and growth.

To define the role of artemin in sympathetic neurone development, we have studied the effect of artemin on the generation, survival and growth of sympathetic neurones in low-density dissociated cultures of mouse cervical and thoracic paravertebral sympathetic ganglia at stages throughout embryonic and postnatal development. Artemin promoted the proliferation of sympathetic neuroblasts and increased the generation of new neurones in cultures established from E12 to E14 ganglia. Artemin also exerted a transient survival-promoting action on newly generated neurones during these early stages of development. Between E16 and P8, artemin exerted no effect on survival, but by P12, as sympathetic neurones begin to acquire neurotrophic factor independent survival, artemin once again enhanced survival, and by P20 it promoted survival as effectively as nerve growth factor (NGF). During this late period of development, artemin also enhanced the growth of neurites from cultured neurones more effectively than NGF. Confirming the physiological relevance of the mitogenic action of artemin on cultured neuroblasts, there was a marked reduction in the rate of neuroblast proliferation in the sympathetic ganglia of mice lacking the GFRalpha3 subunit of the artemin receptor. These results indicate that artemin exerts several distinct effects on the generation, survival and growth of sympathetic neurones at different stages of development.

Activation of expression of hedgehog target genes in basal cell carcinomas.

Mutations in hedgehog signaling pathway genes, especially PTC1 and SMO, are pivotal to the development of basal cell carcinomas. The study of basal cell carcinoma gene expression not only may elucidate mechanisms by which hedgehog signaling abnormalities produce aberrant tumor cell behavior but also can provide data on in vivo hedgehog target gene control in humans. We have found, in comparison with normal skin, that basal cell carcinomas have increased levels of mRNA for PTC1, GLI1, HIP, WNT2B, and WNT5a; decreased levels of mRNA for c-MYC, c-FOS, and WNT4; and unchanged levels of mRNA for PTC2, GLI2, WNT7B, and BMP2 and 4. These findings suggest that mutations in hedgehog signaling pathway genes may exert both cell autonomous and indirect effects and indicate that basal cell carcinoma tumor cells have a phenotype that at least in some aspects resembles that of epidermal stem cells.

Downregulation of Hedgehog signaling is required for organogenesis of the small intestine in Xenopus.

Hedgehog ligands interact with receptor complexes containing Patched (PTC) and Smoothened (SMO) proteins to regulate many aspects of development. The mutation W535L (SmoM2) in human Smo is associated with basal cell skin cancers, causes constitutive, ligand-independent signaling through the Hedgehog pathway, and provides a powerful means to test effects of unregulated Hedgehog signaling. Expression of SmoM2 in Xenopus embryos leads to developmental anomalies that are consistent with known requirements for regulated Hedgehog signaling in the eye and pancreas. Additionally, it results in failure of midgut epithelial cytodifferentiation and of the intestine to lengthen and coil. The midgut mesenchyme shows increased cell numbers and attenuated expression of the differentiation marker smooth muscle actin. With the exception of the pancreas, differentiation of foregut and hindgut derivatives is unaffected. The intestinal epithelial abnormalities are reproduced in embryos or organ explants treated directly with active recombinant hedgehog protein. Ptc mRNA, a principal target of Hedgehog signaling, is maximally expressed at stages corresponding to the onset of the intestinal defects. In advanced embryos expressing SmoM2, Ptc expression is remarkably confined to the intestinal wall. Considered together, these findings suggest that the splanchnic mesoderm responds to endodermal Hedgehog signals by inhibiting the transition of midgut endoderm into intestinal epithelium and that attenuation of this feedback is required for normal development of the vertebrate intestine.

Development of Th1-type immune responses requires the type I cytokine receptor TCCR.

On antigen challenge, T-helper cells differentiate into two functionally distinct subsets, Th1 and Th2, characterized by the different effector cytokines that they secrete. Th1 cells produce interleukin (IL)-2, interferon-gamma (IFN-gamma) and lymphotoxin-beta, which mediate pro-inflammatory functions critical for the development of cell-mediated immune responses, whereas Th2 cells secrete cytokines such as IL-4, IL-5 and IL-10 that enhance humoral immunity. This process of T-helper cell differentiation is tightly regulated by cytokines. Here we report a new member of the type I cytokine receptor family, designated T-cell cytokine receptor (TCCR). When challenged in vivo with protein antigen, TCCR-deficient mice had impaired Th1 response as measured by IFN-gamma production. TCCR-deficient mice also had increased susceptibility to infection with an intracellular pathogen, Listeria monocytogenes. In addition, levels of antigen-specific immunoglobulin-gamma2a, which are dependent on Th1 cells, were markedly reduced in these mice. Our results demonstrate the existence of a new cytokine receptor involved in regulating the adaptive immune response and critical to the generation of a Th1 response.

Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Smoothened activates Galphai-mediated signaling in frog melanophores.

The 7-pass transmembrane protein Smoothened was investigated for its ability to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into immortalized frog pigment cells. Cells expressing the protein showed a phenotype of persistent pigment aggregation, a hallmark of constitutive Galpha(i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative Galpha(i). The ability of melanophores to express functional Smoothened was also determined by its co-expression with the twelve-pass transmembrane protein, Patched. Patched blocked Smoothened-mediated melanosome aggregation in a dose-dependent manner, consistent with its physiological role as an inhibitor of Smoothened. That the reconstituted Patched-Smoothened receptor complex functions normally in pigment cells was demonstrated by co-transfection with the activating ligand, Sonic hedgehog, as well as by direct application of the recombinant Sonic hedgehog protein. Sonic hedgehog reversed Patched-mediated inhibition of Smoothened and induced pigment aggregation. The findings demonstrate that the human Sonic hedgehog receptor complex can be functionally reconstituted in melanophores and that it is capable of transmembrane signaling by utilizing endogenous Galpha(i).

Embryonic stem cell differentiation to hematopoietic cells: A model to study the function of various regions of the intracytoplasmic domain of cytokine receptors in vitro.

To examine whether the in vitro model of embryonic stem (ES) cell hematopoietic differentiation is suitable to study the function of intracytoplasmic regions of cytokine receptors, we used the thrombopoietin receptor Mpl as a typical cytokine receptor.ES cells deficient in c-mpl (mpl(-/)-) were transfected with genes encoding the full-length or two mutated forms of the intracytoplasmic domain of Mpl using the pEF-BOS expression vector. The mutated forms lack box1 or box2.pEF-BOS was able to maintain protein production during ES cell differentiation. Reintroduction of full-length-c-mpl into mpl(-/)- ES cells restored the response of megakaryocyte progenitors to a truncated form of human Mpl-ligand conjugated to polyethylene glycol (PEG-rhuMGDF) and the formation of platelets, for which mpl(-/)- ES cells are defective. In addition, enforced expression of Mpl resulted in the development of all myeloid progenitors and mature cells in the presence of PEG-rhuMGDF. Blast colony-forming cells, the in vitro equivalent of the hemangioblast, also generated blast cell colonies with a hematopoietic potential equivalent to that of the wild type in the presence of PEG-rhuMGDF, although its growth is normally dependent on vascular endothelial cell growth factor (VEGF). Thus, Mpl acts as a substitute for other cytokine receptors and for a tyrosine kinase receptor, Flk-1, indicating that Mpl has no instructive role in hematopoietic cell commitment and differentiation. The Mpl mutant forms lacking box1 or box2 prevented response of ES cell-derived blast colony-forming cells or progenitors to PEG-rhuMGDF. Therefore, these two regions, essential for signaling by cytokine receptors, are required for the responses of ES cell-derived hematopoietic cells to PEG-rhuMGDF.These results show that the in vitro hematopoietic differentiation of ES cells is suitable for studying the role of various intracytoplasmic regions of cytokine receptors.

Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo.

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Delta60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.

Hedgehog signal transduction: from flies to vertebrates.

The patterning and morphogenesis of multicellular organisms require a complex interplay of inductive signals which control proliferation, growth arrest, and differentiation of different cell types. A number of such signaling molecules have been identified in vertebrates and invertebrates. The molecular dissection of these pathways demonstrated that in vertebrates, mutations or abnormals function of these signaling pathways were often associated with developmental disorders and cancer formation. The Hedgehog (Hh) family of secreted proteins provides a perfect example of such signaling proteins. In the following review, we will not discuss in detail the role of Hh as a morphogen, but rather focus on its signal transduction pathway and its role in various human disorders.

Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli.

Drosophila Suppressor of fused (Su(fu)) encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Here we describe the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) (hSu(fu)). Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms were expressed in multiple embryonic and adult tissues, and exhibited a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus mapped to chromosome 10q24-q25, a region which is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) was found to repress activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homologue. Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway.

Sonic hedgehog signaling by the patched-smoothened receptor complex.

BACKGROUND: The Hedgehog (Hh) family of secreted proteins is involved in a number of developmental processes as well as in cancer. Genetic and biochemical data suggest that the Sonic hedgehog (Shh) receptor is composed of at least two proteins: the tumor suppressor protein Patched (Ptc) and the seven-transmembrane protein Smoothened (Smo). RESULTS: Using a biochemical assay for activation of the transcription factor Gli, a downstream component of the Hh pathway, we show here that Smo functions as the signaling component of the Shh receptor, and that this activity can be blocked by Ptc. The inhibition of Smo by Ptc can be relieved by the addition of Shh. Furthermore, oncogenic forms of Smo are insensitive to Ptc repression in this assay. Mapping of the Smo domains required for binding to Ptc and for signaling revealed that the Smo-Ptc interaction involves mainly the amino terminus of Smo, and that the third intracellular loop and the seventh transmembrane domain are required for signaling. CONCLUSIONS: These data demonstrate that Smo is the signaling component of a multicomponent Hh receptor complex and that Ptc is a ligand-regulated inhibitor of Smo. Different domains of Smo are involved in Ptc binding and activation of a Gli reporter construct. The latter requires the third intracellular loop and the seventh transmembrane domain of Smo, regions often involved in coupling to G proteins. No changes in the levels of cyclic AMP or calcium associated with such pathways could be detected following receptor activation, however.

Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo.

The question of whether extracellular signals influence hematopoiesis by instructing stem cells to commit to a specific hematopoietic lineage (instructive model) or solely by permitting the survival and proliferation of predetermined progenitors (permissive model) has been controversial since the discovery of lineage-dominant hematopoietic cytokines. To study the potential role of cytokines and their receptors in hematopoietic cell fate decisions, we used homologous recombination to replace the thrombopoietin receptor gene (mpl) with a chimeric construct encoding the extracellular domain of mpl and the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This chimeric receptor binds thrombopoietin but signals through the G-CSFR intracellular domain. We found that, despite the absence of a functional mpl signaling domain, homozygous knock-in mice had a normal platelet count, indicating that in vivo the cytoplasmic domain of G-CSFR can functionally replace mpl signaling to support normal megakaryopoiesis and platelet formation. This finding is compatible with the permissive model, according to which cytokine receptors provide a nonspecific survival or proliferation signal, and argues against an instructive role of mpl or G-CSFR in hematopoietic cell fate decisions.

Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear.

The cochlea and vestibular structures of the inner ear labyrinth develop from the otic capsule via step-wise regional and cell fate specification. Each inner ear structure contains a sensory epithelium, composed of hair cells, the mechanosensory transducers, and supporting cells. We examined the spatio-temporal expression of genes in the Notch signaling pathway, Notch receptors (Notch1-4) and two ligands, Jagged1 and Delta1, in the developing mammalian inner ear. Our results show that Notch1 and Jagged1 are first expressed in the otic vesicle, likely involved in differentiation of the VIIIth nerve ganglion neurons, and subsequently within the inner ear sensory epithelia, temporally coincident with initial hair cell differentiation. Notch1 expression is specific to hair cells and Jagged1 to supporting cells. Their expression persists into adult. Notch2, Notch3, Notch4, and Delta1 are excluded from the inner ear epithelia. These data support the hypothesis that Notch signaling is involved in hair cell differentiation during inner ear morphogenesis.

Characterization of two patched receptors for the vertebrate hedgehog protein family.

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human PTCH2 gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.

Role of c-mpl in early hematopoiesis.

Recently, several lines of evidence have indicated an expanded role for thrombopoietin (TPO) and its receptor, c-mpl, in hematopoiesis. In addition to being the primary physiological regulator of platelet production, it is now apparent that TPO also acts during early hematopoiesis. To futher define the role of TPO in early hematopoiesis we have identified discrete murine and human stem cell populations with respect to c-mpl expression and evaluated their potential for hematopoietic engraftment. Fluorescence-activated cell sorter analysis of enriched stem cell populations showed the presence of c-mpl expressing subpopulations. Approximately 50% of the murine fetal liver stem cell-enriched population, AA4(+)Sca+c-kit+, expressed c-mpl. Analysis of the murine marrow stem cell population LinloSca+c-kit+ showed that 70% of this population expressed c-mpl. Expression of c-mpl was also detected within the human bone marrow CD34(+)CD38(-) stem cell progenitor pool and approximately 70% of that population expressed c-mpl. To rigorously evaluate the role of TPO/c-mpl in early hematopoiesis we compared the repopulation capacity of murine stem cell populations with respect to c-mpl expression in a competitive repopulation assay. When comparing the fetal liver progenitor populations, AA4(+)Sca+c-kit+c-mpl+ and AA4(+)Sca+c-kit+c-mpl-, we found that stem cell activity segregates with c-mpl expression. This result is complemented by the observation that the LinloSca+ population of c-mpl gene-deficient mice was sevenfold less potent than LinloSca+ cells from wild-type mice in repopulating activity. The engraftment potential of the human CD34(+)CD38(-)c-mpl+ population was evaluated in a severe combined immunodeficient-human bone model. In comparison to the CD34(+) CD38(-)c-mpl- population, the CD34(+)CD38(-)c-mpl+ cells showed significantly better engraftment. These results demonstrate a physiological role for TPO and its receptor, c-mpl, in regulating early hematopoiesis.

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.