Patched Research Articles

PTCH mutations in four Brazilian patients with holoprosencephaly and in one with holoprosencephaly‐like features and normal MRI

We report five Brazilian probands with PATCHED (PTCH) mutations and highly variable phenotypes with holoprosencephaly in four cases and holoprosencephaly-like facial features with a normal MRI in a fifth case. Three of our mutations were novel: Ala443Gly, Val751Gly, and Val908Gly. Two patients had the same mutation (Val908Gly), but were phenotypically different: alobar holoprosencephaly, absent nasal septum, and midline cleft lip-palate in one case, and lobar holoprosencephaly, macrocephaly, hypertelorism, clefting of the nose, severe microphthalmia, and a single maxillary central incisor in the other. One of our patients had a Thr1052Met mutation, holoprosencephaly-like facial features, and a normal MRI. Ming et al. [(2002); Hum Genet 110:297-301] reported an identical mutation, but with alobar holoprosencephaly.

Patched mutations and hairy skin patches: A new sign in Gorlin syndrome

We report on the occurrence of discrete patches of unusually long pigmented hair on the skin of three patients with Gorlin syndrome from two unrelated families with confirmed heterozygous mutations in the Patched (PTCH) gene. The PTCH protein is a negative regulator of Hedgehog signaling, and the Sonic Hedgehog (SHH)-PTCH pathway is known to play an important role in the formation and cycling of the hair follicle. We believe that the patches represent a genuine physical sign associated with Gorlin syndrome, and discuss molecular mechanisms by which they might arise.

PTCH mutations and deletions in patients with typical nevoid basal cell carcinoma syndrome and in patients with a suspected genetic predisposition to basal cell carcinoma: a French study

The patched (PTCH) mutation rate in nevoid basal cell carcinoma syndrome (NBCCS) reported in various studies ranges from 40 to 80%. However, few studies have investigated the role of PTCH in clinical conditions suggesting an inherited predisposition to basal cell carcinoma (BCC), although it has been suggested that PTCH polymorphisms could predispose to multiple BCC (MBCC). In this study, we therefore performed an exhaustive analysis of PTCH (mutations detection and deletion analysis) in 17 patients with the full complement of criteria for NBCCS (14 sporadic and three familial cases), and in 48 patients suspected of having a genetic predisposition to BCC (MBCC and/or age at diagnosis ⩽40 years and/or familial BCC). Eleven new germline alterations of the PTCH gene were characterised in 12 out of 17 patients harbouring the full complement of criteria for the syndrome (70%). These were frameshift mutations in five patients, nonsense mutations in five patients, a small inframe deletion in one patient, and a large germline deletion in another patient. Only one missense mutation (G774R) was found, and this was in a patient affected with MBCC, but without any other NBCCS criterion. We therefore suggest that patients harbouring the full complement of NBCCS criteria should as a priority be screened for PTCH mutations by sequencing, followed by a deletion analysis if no mutation is detected. In other clinical situations that suggest genetic predisposition to BCC, germline mutations of PTCH are not common.

Hedgehog signalling in skin development and cancer

Basal cell carcinoma (BCC) is the most common human malignancy, affecting 750,000 Americans each year. The understanding of mutations that are known to activate hedgehog (Hh) signalling pathway genes, including PATCHED (PTCH), sonic hedgehog (Shh) and smoothened (Smo), has substantially expanded our current understanding of the genetic basis of BCC development. The Hh signalling pathway is one of the most fundamental signal transduction pathways in embryonic development. In skin, the Shh pathway is crucial for maintaining stem cell population, and for regulating hair follicle and sebaceous gland development. This pathway plays a minimal role in adult tissues, but is known to be activated in many neoplasms, including those arising in the skin. In this review, we attempt to summarize the results of published studies on some important aspects of the Shh pathway and its involvement in skin development and carcinogenesis. We also provide a description of various animal models that have been developed, based on our knowledge of the Shh pathway in human skin cancers. Additionally, we include a brief description of studies conducted in our laboratory and by others on the chemoprevention of BCCs. This review therefore provides a current understanding of the role of the Shh pathway in skin development and neoplasia. It also provides a basis for the molecular target-based chemoprevention and therapeutic management of skin cancer.

MC1R and PTCH Gene Polymorphism in French Patients with Basal Cell Carcinomas

In this study, we assessed the role of melanocortin 1 receptor (MC1R) variants and of two patched (PTCH) polymorphisms (c.3944C>T (P1315L), insertion 18 bp IVS1-83) as risk factors for basal cell carcinoma (BCC) in the French population. The population investigated comprised 126 BCC patients who were enrolled on the basis of specific criteria (multiple and/or familial BCC and/or onset before the age of 40 years and/or association with another tumor)--and 151 controls matched for ethnicity, age, and sex. MC1R variants appeared as a moderate risk factor for BCC (odds ratio (OR) for one and two variants, 2.17 [1.28-3.68] and 7.72 [3.42-17.38], respectively), independently of pigmentation characteristics (OR = 2.53 [1.34-4.8]). Interestingly, in addition to the predictable red hair color (RHC) alleles, two non-RHC alleles (V60L and V92M) were also closely associated with BCC risk (OR 3.21 [1.91-5.38] and 2.87 [1.5-5.48], respectively), which differs from the situation in the Celtic population. In addition, the PTCH c.3944C/C genotype was also associated with BCC risk (OR 1.94 [1.2-3.1]), especially in the subgroup of patients with multiple tumors (OR 2.16 [1.3-3.6]). Thus, our data show that MC1R and PTCH variants are associated with BCC risk in the French population. We further suggest that assessing MC1R and PTCH status could be useful, combined with the assessment of clinical risk factors, in identifying high-risk patients to be targeted for prevention or more rigorous surveillance.

Hedgehog Signal Activation in Gastric Pit Cell and in Diffuse-Type Gastric Cancer

Sonic hedgehog (SHH) and Indian hedgehog (IHH) have cell-specific actions in some organs. Only SHH has been shown to regulate parietal cell differentiation. This study examined whether SHH, 2 other ligands IHH and Desert hedgehog, and receptors or downstream targets are expressed in normal gastric epithelium or in intestinal and diffuse-type gastric cancers. The effects of a Hedgehog (Hh) inhibitor, cyclopamine, were assessed in primary gastric epithelium cultures and gastric cancer cell lines. Reverse-transcription polymerase chain reaction and immunostaining compared expression and localization of Hh signaling molecules and phenotypic markers of pit, neck, and gland cells in situ and in cultured cells treated with cyclopamine. Bromodeoxyuridine staining assessed the effects of cyclopamine on proliferation. Hh signaling molecules were expressed differentially in pit, neck, and gland cells. IHH co-expressed with most downstream targets in the pit. IHH, SHH, Patched (PTCH), Smoothened (SMO), and downstream targets were expressed more frequently and highly in the diffuse as compared with intestinal type cancers. In diffuse cancers, IHH was expressed in cells with an epithelial phenotype and SHH in cells with a mesenchymal phenotype. Cyclopamine reduced the number of cells with a pit phenotype but not a gland phenotype in primary cultures. Cyclopamine had particularly potent effects of inhibiting the growth of cell lines that expressed high levels of SMO. Expression of IHH and downstream targets correlates with pit cells. IHH and SMO may be useful biomarkers of diffuse cancers that may show growth inhibition with Hh antagonists such as cyclopamine.

Peripheral chondrosarcoma progression is accompanied by decreased Indian Hedgehog signalling

Hedgehog (HH) signalling is important for specific developmental processes, and aberrant, increased activity has been described in various tumours. Disturbed HH signalling has also been implicated in the hereditary syndrome, Multiple Osteochondromas. Indian Hedgehog (IHH), together with parathyroid hormone-like hormone (PTHLH), participates in the organization of growth plates in long bones. PTHLH signalling is absent in osteochondromas, benign tumours arising adjacent to the growth plate, but is reactivated when these tumours undergo malignant transformation towards secondary peripheral chondrosarcoma. We describe a gradual decrease in the expression of Patched (PTCH) and glioma-associated oncogene homologue 1 (GLI1) (both transcribed upon IHH activity), and GLI2 with increasing malignancy, suggesting that IHH signalling is inactive and PTHLH signalling is IHH independent in secondary peripheral chondrosarcomas. cDNA expression profiling and immunohistochemical studies suggest that transforming growth factor-beta (TGF-beta)-mediated proliferative signalling is active in high-grade chondrosarcomas since TGF-beta downstream targets were upregulated in these tumours. This is accompanied by downregulation of energy metabolism-related genes and upregulation of the proto-oncogene jun B. Thus, the tight regulation of growth plate organization by IHH signalling is still seen in osteochondroma, but gradually lost during malignant transformation to secondary peripheral chondrosarcoma and subsequent progression. TGF-beta signalling is stimulated during secondary peripheral chondrosarcoma progression and could potentially regulate the retained activity of PTHLH.

Hedgehog signaling and response to cyclopamine differs in epithelial and stromal cells in benign breast and breast cancer

The hedgehog pathway regulates epithelial-mesenchymal interactions, differentiation, proliferation and survival during development. Stimulation of hedgehog signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. Using real-time, quantitative PCR, laser capture microdissection, and immunohistochemistry, distinctive patterns of expression of the hedgehog pathway members patched 1 (PTCH1), smoothened, GLI1, GLI2 and the 3 hedgehog ligands were identified for epithelial cells and stromal fibroblasts in benign breast and breast cancer. Hedgehog ligands were expressed at higher levels in some cancer epithelial cell lines compared to non-cancerous epithelial cells. Correspondingly, expression of GLI1, a transcription factor and transcriptional product of hedgehog signaling, was increased 8-fold in cancer epithelial cell lines; however, PTCH1, also a transcriptional target of hedgehog signaling in many cell types, was not increased. GLI1 protein and mRNA, and PTCH1 and sonic hedgehog (SHH) proteins were elevated in 3 of 10 breast cancers; however, PTCH1 transcripts were not consistently increased. Hedgehog-mediated transcription, as indicated by a reporter of GLI-dependent promoter activity and by expression of GLI1 transcripts, was reduced by the hedgehog pathway inhibitor cyclopamine in both MDA-MB-435 cancer epithelial cells and MCF10AT epithelial cells, a cell line derived from benign breast. However, cyclopamine reduced viability of cancer epithelial cell lines, including MDA-MB-435, but did not specifically affect fibroblasts or epithelial cells from benign breast, including MCF10AT. Treatment with sonic hedgehog ligand diminished the cyclopamine-induced reduction in GLI-dependent promoter activity in MCF10AT and MDA-MB-435 and viability of MDA-MB-435. These results demonstrate modulation of GLI-mediated transcription in both cancer and benign-derived epithelial cells by cyclopamine and sonic hedgehog, and further suggest that hedgehog signaling contributes to the survival of only the cancer epithelial cells. Determination as to whether the increase in GLI1 and SHH expression in breast cancer indicates a significant increase in hedgehog signaling will require further evaluation.

Inhibition of GLI1 gene activation by Patched1

Patched1 (PTCH1) is a human tumour suppressor that acts as an HH (Hedgehog) receptor protein and is important for embryonic patterning. PTCH1 mediates its effects through SMO (Smoothened) and represses the expression of HH target genes such as the transcription factor GLI1 (glioma 1) as well as PTCH1. Up-regulation of these genes has been observed in several cancer forms, including basal cell carcinoma, digestive track tumours and small cell lung cancer. The fact that PTCH1 down-regulates its own expression via 'negative feedback' is an important feature in HH signalling, as it keeps the balance between HH and PTCH1 activities that are essential for normal development. In the present study, we provide evidence that a novel mechanism allowing PTCH1 to maintain this balance may also exist. We show that gene activation by GLI1, the transcriptional effector of the pathway, can be down-regulated by PTCH1 without involvement of the canonical cascade of HH signalling events. Specifically, the SMO antagonist cyclopamine has no appreciable effects in blocking this PTCH1-mediated inhibition. Moreover, the negative GLI1 regulator SUFU (Suppressor of Fused) was also found to be dispensable. Additionally, deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180-786 and 1058-1210 are of highest significance in inhibiting GLI1 gene activation. This contrasts with the importance of the PTCH1 C-terminal domain for HH signalling.

Smoothened overexpression activates hedgehog signaling in human hepatocarcinogenesis

BACKGROUND: Hedgehog pathway activation promotes tumors in several endodermally-derived tissues including the pancreas and bile ducts, but its role in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. Although normal hepatocytes lack Hedgehog signaling, activation of the Hedgehog pathway in endodermal progenitors is required for liver development. Hedgehog signaling is activated when Hedgehog ligands (Sonic hedgehog or Indian hedgehog) bind to their receptor, Patched (PTC). In an unoccupied state, PTC functions as a tumor suppressor that inhibits Smoothened (SMO), a proto-oncoprotein, from inducing downstream signaling and transcription of target genes.

Contributions of PTCH gene variants to isolated cleft lip and palate.

Mutations in patched (PTCH) cause the nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin syndrome. Nevoid basal cell carcinoma syndrome may present with developmental anomalies, including rib and craniofacial abnormalities, and predisposes to several tumor types, including basal cell carcinoma and medulloblastoma. Cleft palate is found in 4% of individuals with nevoid basal cell carcinoma syndrome. Because there might be specific sequence alterations in PTCH that limit expression to orofacial clefting, a genetic study of PTCH was undertaken in cases with cleft lip and/or palate (CL/P) known not to have nevoid basal cell carcinoma syndrome. Seven new normal variants spread along the entire gene and three missense mutations were found among cases with cleft lip and/or palate. One of these variants (P295S) was not found in any of 1188 control samples. A second variant was found in a case and also in 1 of 1119 controls. The third missense (S827G) was found in 5 of 1369 cases and in 5 of 1104 controls and is likely a rare normal variant. Linkage and linkage desequilibrium also was assessed using normal variants in and adjacent to the PTCH gene in 220 families (1776 individuals), each with two or more individuals with isolated clefting. Although no statistically significant evidence of linkage (multipoint HLOD peak = 2.36) was uncovered, there was borderline evidence of significant transmission distortion for one haplotype of two single nucleotide polymorphisms located within the PTCH gene (p = .08). Missense mutations in PTCH may be rare causes of isolated cleft lip and/or palate. An as yet unidentified variant near PTCH may act as a modifier of cleft lip and/or palate.

Inhibition of SHH signaling pathway: Molecular treatment strategy of odontogenic keratocyst

Odontogenic keratocyst (OKC) is a relatively common cystic lesion occurred in the tooth-bearing areas of the jaws. This entity is thought to arise from the dental lamina or its remnant with significant growth capacity and recurrence potential. The Sonic hedgehog (SHH) signaling pathway plays a critical role in tooth development. Patched (PTCH) combines with Smoothened (SMO) to form a receptor complex for SHH ligand. Mutations in the PTCH resulting in aberrant activation of SHH signaling pathway were identified as the underlying genetic event of both sporadic and syndrome-related OKCs. We postulate that any strategy to develop antagonists of active receptor, transcriptional factors of SHH signaling pathway will be an effective treatment for OKC. These strategies include reintroducing a wild-type form of PTCH, inhibition of the SMO molecule by synthetic small antagonists and suppression of the downstream transcription factors of the SHH signaling pathway. It seems that inhibition of SMO by intracystic injection of antagonist protein of SMO is the most potential treatment choice.

Enhanced expression of hedgehog signaling molecules in squamous cell carcinoma of uterine cervix and its precursor lesions

The hedgehog (Hh)-signaling pathway plays an essential role in normal development. Deregulation of this pathway is responsible for several types of cancers. The aim of this study was to determine the expression pattern and the extent of Hh-signaling molecules in squamous cell carcinoma of uterine cervix and its precursor lesions. A total of 106 uterine cervical cancers and related lesions (37 squamous cell carcinomas, 23 cervical intraepithelial neoplasia (CIN) III, 10 CIN II, four CIN I, 32 normal cervical epithelia) were immunohistochemically analyzed with anti-Shh, Indian Hh (Ihh), Patched (PTCH), Smoothened (Smo), Gli-1, Gli-2, Gli-3 antibodies on paraffin blocks. The results showed that the expression of all the Hh-signaling molecules was greatly enhanced in uterine cervical tumors, including carcinoma and its precursor lesions. The staining pattern was mainly cytoplasmic except for Gli-1/2, whose expression was observed in both cytoplasm and nucleus. In case of Ihh, PTCH, Smo and Gli-1, their expression in normal epithelium was completely absent or rare. The expression of all the seven Hh-signaling molecules mentioned above was significantly increased in CIN II/III and carcinoma, compared with that in normal epithelium (P<0.05). The expression of Shh was increased by double; the first increase occurred in normal epithelium–CIN transition, and the second, during the progression of CIN to carcinoma. These results strongly suggest that the Hh-signaling pathways were extensively activated in carcinoma and CIN of uterine cervix. In conclusion, the Hh-signaling pathways may be involved in carcinogenesis of squamous cell carcinoma of uterine cervix and can be considered as a potential therapeutic target.

Dysregulation of the Hedgehog pathway in human hepatocarcinogenesis

Hedgehog (Hh) pathway activation promotes tumors in several endodermally derived tissues, but its role in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. Although normal hepatocytes lack Hh signaling, activation of the Hh pathway in endodermal progenitors is required for liver development. Thus, we hypothesized that hepatocarcinogenesis may involve regulation of Hh signaling. This pathway is activated when Hh ligand binds to its receptor, Patched (PTC). In an unoccupied state, PTC normally functions as a tumor suppressor that inhibits Smoothened (SMO), a proto-oncoprotein, from activating downstream components and transcription of target genes. Here we show that in HCCs, overexpression of the Smo proto-oncogene, as well as an increase in the stoichiometric ratio of Smo to Ptc mRNA levels, correlated with tumor size, a prognostic indicator in HCC biology. In one tumor we identified a novel Smo mutation in an evolutionarily conserved residue. We also demonstrated that HCC cell lines (HepG2 and Hep3B) expressed Hh pathway components and activated Hh transcriptional targets. In Hep3B cells, cyclopamine, an inhibitor of wild-type SMO, had no effect, but KAAD-cyclopamine, a blocker of oncogenic SMO, inhibited Hh signaling activity by 50%, decreased expression of the hepatocarcinogenic oncogene, c-myc, by 8-fold, and inhibited the growth rate of Hep3B cells by 94%. These data support our hypothesis that Hh signaling is dysregulated in human hepatocarcinogenesis. We demonstrate that overexpression and/or tumorigenic activation of the Smo proto-oncogene mediates c-myc overexpression which plays a critical role in hepatocarcinogenesis and suggests that Smo is a prognostic factor in HCC tumorigenesis.

Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays

Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

The elusive multiple self-healing squamous epithelioma (MSSE) gene: further mapping, analysis of candidates, and loss of heterozygosity

The MSSE gene predisposes to multiple invasive but self-healing skin tumours (multiple self-healing epitheliomata). MSSE was previously mapped to chromosome 9q22-q31 and a shared haplotype in affected families suggested a founder mutation. We have refined the MSSE critical region (<1 cM, <1 Mb) between the zinc-finger gene ZNF169 and the Fanconi anaemia gene FANCC. By genetic mapping we have excluded ZNF169 and FANCC as well as PTCH (PATCHED) and TGFBR1 (transforming growth factor beta receptor type-1) genes. The CDC14B cell cycle phosphatase gene also lies in the region but screening of the complete coding region revealed no mutation in MSSE patients. Somatic cell hybrids created by haploid conversion of an MSSE patient's cells enabled screening of the MSSE chromosome 9 and showed no CDC14B deletion or mutation that abrogates CDC14B mRNA expression. Thus, CDC14B is unlikely to be the MSSE gene. We also report the first molecular analysis of MSSE tumours showing loss of heterozygosity of the MSSE region, with loss of the normal allele, providing the first evidence that MSSE is a tumour suppressor gene.

Gli1 is important for medulloblastoma formation in Ptc1+/− mice

Germline mutations in the human homolog of the patched1 (PTCH1) are associated with basal cell nevus carcinoma syndrome (BCNS or Gorlin syndrome), which is characterized by developmental anomalies, radiation hypersensitivity and a predisposition to medulloblastomas and skin tumors. Patched1 (Ptc1) functions as a receptor for Sonic hedgehog (Shh) in a wide range of biological processes. Binding of Shh to Ptc1 results in activation of Smoothened (Smo), which in turn stimulates expression of downstream target genes including Ptc1 and Gli1. Gli1 is a member of a family of DNA-binding zinc-finger proteins, including Gli2 and Gli3, that function in transcription control. Here, we report that inactivation of both Gli1 alleles in Ptc1+/- mice significantly reduces spontaneous medulloblastoma formation. Therefore, Gli1 is not only a marker of pathway activation but also plays a functional role in medulloblastoma formation. Interestingly, Gli2 levels were elevated in medulloblastoma cells but not in normal granule neuron precursors during cerebellar development in mice lacking Gli1. In cultured fibroblasts, Gli1 was more potent than Gli2 at inducing cell transformation. These results demonstrate that Gli1 plays a central role in medulloblastoma formation in Ptc1+/- mice and that Gli2 may also contribute to oncogenesis.

Retrospective family study of childhood medulloblastoma

Medulloblastoma is the most common malignant central nervous system tumor of childhood and can occur sporadically or in association with inherited cancer susceptibility syndromes such as the nevoid basal cell carcinoma syndrome (NBCCS). To determine whether an association existed between the risk of developing medulloblastoma and undiagnosed syndromes, we retrospectively reviewed clinical data on 33 patients with medulloblastoma from a single institution and compared them with their unaffected relatives (n = 46). Six patients had tumors showing desmoplastic histology. Two of the six met diagnostic criteria for NBCCS. One NBCCS patient had a missense mutation of patched-1 (PTCH1); the other had no identifiable PTCH1 mutation. Two patients with isolated desmoplastic medulloblastoma had an insertion and splice site mutation, respectively, in suppressor of fused (SUFU). All patients with nondesmoplastic medulloblastoma histology received molecular testing for SUFU. None of these patients had an identifiable mutation in PTCH1 or SUFU. We performed a clinical evaluation for Greig cephalopolysyndactyly syndrome (GCPS) in four medulloblastoma families, who exhibited macrocephaly as the only finding consistent with the diagnosis of GCPS. Molecular analysis of GLI3 in these four families was negative. There was a paucity of clinical findings among the majority of medulloblastoma patients in this study group to suggest a definable cancer genetic syndrome. We conclude that clinically recognizable syndromes are uncommon among patients with medulloblastoma, however, PTCH1 and SUFU mutations are present at a low but significant frequency.

Functions of Sorting Nexin 17 Domains and Recognition Motif for P-selectin Trafficking

SNX17 is a member of the sorting nexin family (SNX), a group of hydrophilic proteins whose common characteristic property is a phox homology (PX) domain. The PX domain directs SNXs to phosphatidylinositides containing membranes of the endosomal compartment, where the SNXs are involved in the sorting of transmembrane proteins. SNX17 is known to interact with P-selectin and the LDL receptor family. Here, we report that the PX domain of SNX17 specifically binds to phosphatidylinositol 3-phosphate-containing membranes. The functional part of SNX17 that binds P-selectin or Patched (PTCH) consists of a truncated FERM domain and a unique C terminus together (FC-unit). In a yeast two-hybrid analysis a putative recognition motif for the FC-unit was revealed within P-selectin as FxNaa(F/Y). When HepG2 cells overexpress P-selectin together with SNX17, SNX17 changes its distribution from early endosomes to lysobisphosphatidic acid-containing late endosomes. Furthermore, overexpressed SNX17 restrains P-selectin in the outer membrane of the late endosomal compartment, thus preventing the normal lysosomal accumulation of P-selectin. These results suggest that the PX domain is necessary for the intracellular localisation, while the FC-unit is required for cargo recognition. We hypothesise that the expression level of SNX17 may regulate the lysosomal degradation, at least for P-selectin, by suppressing its entry into the inner vesicles of the multi-vesicular bodies (MVBs).

Identification and characterization of multiple isoforms of a murine and human tumor suppressor, patched, having distinct first exons

Mutations in mouse and human patched ( PTCH) genes are associated with birth defects and cancer. PTCH, a 12-pass transmembrane protein, is a receptor for Sonic hedgehog (Shh) signaling proteins. Shh proteins activate transcription of target genes, including PTCH, via GLI transcription factors. Here we identified seven and five isoforms of human and mouse PTCH mRNA, respectively, which are generated by the complex alternative use of five exons as the first exon (exons 1a to 1e in the 5′-to-3′ order). Although expression profiles of these isoforms were highly variable among human tissues, three of them, PTCHa, PTCHb, and PTCHd, were predominantly expressed in most tissues, PTCHd being most ubiquitous. In contrast, PTCHb was always predominant and reached a maximum at E10.5 during mouse development. These three mRNA isoforms encode three PTCH proteins with distinct N-termini, PTCH L, PTCH M, and PTCH S. The expression of these three isoforms was regulated by GLI transcription factors, and at least two functional GLI-binding sequences were identified, one in exon 1a and the other between exon 1a and exon 1b. PTCH L and PTCH M were equally active in terms of suppressing GLI-mediated transcription and inducing apoptosis. PTCH S protein (encoded by PTCHd), lacking the first transmembrane domain, was more unstable than the other two, resulting in a reduced activity. This study may shed light on the mechanism whereby a single PTCH gene plays a role in both tumor cell growth and embryonic development.