Biomedical subjects

Rune Toftgård

Publications and source records attributed to Rune Toftgård.

15 recordsLinked to original sources

PubMed2006

Epidermal and craniofacial defects in mice overexpressing Klf5 in the basal layer of the epidermis.

Krüppel-like factor5 (Klf5) is a zinc-finger transcription factor normally expressed in the skin. Here, we show that overexpression of Klf5 in the basal layer of the epidermis during embryogenesis affects epidermal development and disrupts epithelial-mesenchymal interactions necessary for skin adnexae formation as well as craniofacial morphogenesis. The transgenic mice exhibited exencephaly, craniofacial defects, persistent abdominal herniation and ectodermal dysplasia. Moreover, the epidermis was hypoplastic and underwent abnormal differentiation with expression of keratin8, a marker for single-layered epithelia, in the stratified epidermis. Correspondingly, we observed a downregulation of DeltaNp63 expression in the skin. Overexpression of Klf5 in adult mice led to hyperkeratosis, follicle occlusion and epidermal erosions. Further, we observed decrease and even loss of the stem cell population of bulge keratinocytes, as characterized by the expression pattern of alpha6 integrin and CD34 markers. Our data suggest a new role of Klf5 as a modulator of p63 expression and the differentiation program of epidermal cells relevant for regenerative potential of the epidermis and epithelial-mesenchymal interactions.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

PTCH mutations: distribution and analyses.

Mutations in the PTCH (PTCH1) gene are the underlying cause of nevoid basal cell carcinoma syndrome (NBCCS), and are also found in many different sporadic tumors in which PTCH is thought to act as a tumor suppressor gene. To investigate the distribution pattern of these mutations in tumors and NBCCS, we analyzed 284 mutations and 48 SNPs located in the PTCH gene that were compiled from our PTCH mutation database. We found that the PTCH mutations were mainly clustered into the predicted two large extracellular loops and the large intracellular loop. The SNPs appeared to be clustered around the sterol sensing domain and the second half of the protein. The NBCCS cases and each class of tumor analyzed revealed a different distribution of the mutations in the various PTCH domains. Moreover, the types of mutations were also unique for the different groups. Finally, the PTCH gene harbors mutational hot spot residues and regions, including a slippage-sensitive sequence in the N-terminus.

Basal Cell Nevus Syndrome↗

PubMed2006

Deregulation of the hedgehog signalling pathway: a possible role for the PTCH and SUFU genes in human rhabdomyoma and rhabdomyosarcoma development.

The naevoid basal cell carcinoma syndrome (NBCCS) is caused by mutations in the hedgehog receptor PTCH gene. It is characterized by developmental defects and a predisposition to the development of certain tumours, such as basal cell carcinoma, medulloblastoma and meningioma, and potentially fetal rhabdomyomas and embryonal rhabdomyosarcomas. This study aimed to analyse PTCH status in an NBCCS patient with fetal rhabdomyoma and to investigate whether deregulation of hedgehog signalling, as shown by altered expression of hedgehog pathway components and/or genetic imbalances, is a general finding in sporadic rhabdomyomas and rhabdomyosarcomas. The NBCCS patient had a novel PTCH germ-line mutation, 1370insT, and developed a fetal rhabdomyoma that harboured a 30 bp in-frame deletion in the second allele resulting in homozygous inactivation of PTCH. Sporadic rhabdomyomas and rhabdomyosarcomas showed overexpression of PTCH (43/43) and GLI1 (41/43) mRNA, as determined by in situ hybridization, indicating ongoing active hedgehog signalling. Immunohistochemical staining revealed a subgroup of fetal rhabdomyomas and embryonal rhabdomyosarcomas (12/34) lacking PTCH immunoreactivity. Four of nine informative fetal rhabdomyomas and embryonal rhabdomyosarcomas showed loss of heterozygosity (LOH) in the PTCH region with two of these (one fetal rhabdomyoma and one embryonal rhabdomyosarcoma) also showing LOH in the SUFU region. These findings suggest that haploinsufficiency for the two tumour suppressor genes PTCH and SUFU, which are both active in the same signalling pathway, may be important for tumour development. Based on our results we propose that the pathogenesis of rhabdomyoblastic tumours, particularly fetal rhabdomyomas and embryonal rhabdomyosarcomas, involves deregulation of the hedgehog signalling pathway.

Adolescent↗

PubMed2006

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.

Animals↗

PubMed2004

The FU gene and its possible protein isoforms.

BACKGROUND: FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. RESULTS: The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. CONCLUSIONS: The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling.

Chromosome Mapping↗

PubMed2004

Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site.

The PTCH1 tumor suppressor gene encodes a receptor for secreted hedgehog (HH) ligands and is important for proper proliferation, differentiation and patterning in almost every tissue and organ during embryogenesis. The PTCH1 protein works as a negative regulator of the HH-signaling pathway by repressing downstream signaling by the coreceptor smoothened (SMOH). Mutations in PTCH1 lead to constitutive expression of HH target genes and a relationship between mutated PTCH1 and the most common tumor form in the Western world, Basal Cell Carcinoma (BCC) has been clearly established. We here show that PTCH1 is transcriptionally regulated by three independent promoters generating transcripts with alternative first exons. We demonstrate that only one of two putative Gli-binding sites that were identified in the promoter region of PTCH1 is functional, and that the transactivating Gli proteins, GLI1, Gli2 and GLI3, bind and enhance transcription through this site. Moreover, a strong repression of both basal and induced PTCH1 transcription was observed following expression of a truncated version of GLI3. Most interestingly, the upstream components in the HH-signaling cascade, Sonic HH (SHH) and SMOH, solely operate through the functional Gli-binding site because mutation of the Gli-binding site resulted in the disappearance of the enhanced transcription induced by the Gli proteins, as well as by SHH or SMOH. This finding suggests that transcriptional activation of the PTCH1 gene mediated via the HH-signaling pathway is dependent on the single functional Gli-binding site.

3T3 Cells↗

PubMed2004

Tumor necrosis factor receptor 1-mediated signaling is required for skin cancer development induced by NF-kappaB inhibition.

NF-kappaB signaling plays an important role in skin development and epidermal growth control. Moreover, inhibition of NF-kappaB signaling in murine epidermal keratinocytes in vivo, by expression of a keratin 5 (K5)-directed superrepressor form of inhibitor of NF-kappaB (IkappaBalpha), results in an inflammatory response characterized by a massive dermal infiltration of neutrophils, epidermal hyperplasia, and a rapid development of aneuploid squamous cell carcinomas (SCC). We now show that by crossing K5-IkappaBalpha mice onto a tumor necrosis factor receptor 1(Tnfr1)-null background, both the inflammatory and the tumorigenic responses are blocked. The specificity of the block is illustrated by the fact that K5-IkappaBalpha mice lacking the IL-1 receptor type 1 (Il1r1) develop inflammation and squamous cell carcinomas. Reconstitution of lethally irradiated K5-IkappaBalpha/Tnfr1(-/-) mice with Tnfr1(+/-) bone-marrow does not induce the inflammatory or the tumorigenic phenotype, indicating a critical dependence on Tnfr1-mediated signaling in skin cells or nonimmune cells. Our results suggest a critical role of local Tnfr1-mediated signaling and associated inflammatory response cooperating with repressed keratinocyte NF-kappaB signaling in driving skin cancer development.

Animals↗

PubMed2004

Involvement of RFX proteins in transcriptional activation from a Ras-responsive enhancer element.

The B10.RRE element has previously been shown to mediate induced transcription in response to an activated Ha- ras gene and epidermal growth factor in keratinocytes but not in fibroblasts. We report the identification of regulatory factor for X box 3 (RFX-3) as a B10.RRE-binding protein, using the yeast one-hybrid assay. We showed that in vitro-translated RFX3, as well as RFX1 and RFX2, was able to bind B10.RRE in a sequence-specific manner. Furthermore, RFX proteins are part of the protein complex in HeLa and HepG2 cells that binds the B10.RRE element. Functional analysis in cell culture demonstrated that a truncated version of RFX1 (*RFX1) lacking the repressor domain was able to activate transcription from B10.RRE. Conversely, a dominant-negative form of RFX1 was able to block Ras-induced transcription. Taken together, these results suggest a novel role for the RFX family of transcription factors as modulators of Ras signalling in epithelial cells. Such an interaction is of potential relevance for cell growth and carcinogenesis in the skin.

Antibodies↗

PubMed2004

Distinct roles of PTCH2 splice variants in Hedgehog signalling.

The human PTCH2 gene is highly similar to PTCH1, a tumour suppressor gene frequently mutated in basal cell carcinoma and several other tumour types. PTCH1 is a transmembrane protein believed to inhibit another transmembrane protein SMO (Smoothened), which mediates HH (Hedgehog) signalling. In this study, we analysed the biological properties of several PTCH2 splice variants. An mRNA form that lacked the last exon was abundantly expressed in all tissues examined, in contrast with the one that included it. Moreover, a transcript lacking exon 9, which is a part of a conserved sterol-sensing domain, was identified in intestine, prostate and cerebellum. In ovary, spleen, testis, cerebellum and skin, an mRNA lacking both exons 9 and 10 could also be observed. The different PTCH2 isoforms localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic HH (Shh-N). Additionally, the PTCH2 gene was found to be a target of HH signalling. PTCH2 promoter regulation assays demonstrated that only one of the PTCH2 variants could inhibit the activity of SHH-N, whereas none was capable of inhibiting the activated form of SMO (SMO-M2) and this contrasts with PTCH1. Despite the fact that the PTCH2 isoforms lacked the ability to inhibit SMO-M2 activity, all PTCH2 variants as well as PTCH1, on co-transfection with Smo, were able to change Smo localization from being largely dispersed in the cytoplasm to the juxtanuclear region. Furthermore, the PTCH2 isoforms and PTCH1 co-localized in doubly transfected cells and an interaction between them was confirmed using immunoprecipitation assays. Using Ptch1-/- mouse cells, it was shown that the PTCH2 variants and PTCH1 differentially act to reconstitute not only the SHH but also the Desert HH-dependent transcriptional response. We conclude that in spite of their structural similarities, the PTCH2 isoforms have distinct functional properties when compared with PTCH1.

Alternative Splicing↗

PubMed2004

Characterization of the progressive skin disease and inflammatory cell infiltrate in mice with inhibited NF-kappaB signaling.

A growth inhibitory role in skin development for the NF-kappaB proteins has been established in recent years. We have previously shown that inhibition of NF-kappaB by overexpression of degradation-resistant IkappaB-alpha in the skin results in the development of squamous cell carcinomas (SCC). In this paper, we characterize the progressive skin disease leading to cancer development in mice with inhibited NF-kappaB signaling in the skin. Increased proliferation and a strong inflammatory response were evident in transgenic skin. A mixed inflammatory cell infiltrate dominated by polymorphonuclear leukocytes was observed in concurrence with an upregulation of the proinflammatory cytokine tumor necrosis factor-alpha. This genetically engineered mouse mutation may be a useful tool to test the efficacy of cytokine therapies for SCC in the future.

Animals↗

PubMed2003

Role for ELOVL3 and fatty acid chain length in development of hair and skin function.

Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss.

Acetyltransferases↗

PubMed2002

Alternative first exons of PTCH1 are differentially regulated in vivo and may confer different functions to the PTCH1 protein.

The PTCH1 gene is a human tumour suppressor gene frequently mutated in basal cell carcinoma (BCC) and several other tumour types. It encodes a receptor for soluble factors of the hedgehog family. Binding of hedgehog to the receptor relieves its inhibitory action on the transmembrane co-receptor Smoh. In this study we describe alternative first exons of the PTCH1 tumour suppressor gene and show that they are differentially regulated in normal tissues, exon 1B being expressed at very low levels and the major mRNA species containing exon 1 or 1A. Exon 1B transcripts were found to be specifically upregulated in nodular BCCs. The different PTCH1 transcripts all encode proteins that interact with Smoh in doubly transfected cells. Furthermore, functional assays demonstrated that whereas all PTCH1 isoforms can inhibit the activity of SHH, only the PTCH1B isoform is capable of fully inhibiting Smoh activity. The results indicate that in tumour cells the PTCH1B promoter is specifically activated and importantly, that the N-terminal part of PTCH1 including exon 1B is required for full inhibition of Smoh signaling but not for physical interaction with Smoh.

Alternative Splicing↗

PubMed2002

Inhibition of Rel/Nuclear Factor-kappaB signaling in skin results in defective DNA damage-induced cell cycle arrest and Ha-ras- and p53-independent tumor development.

In recent years a growth inhibitory role in skin for the Rel/NF-kappaB transcription factors has been established, and the block of Rel/NF-kappaB signaling results in rapid development of spontaneous skin cancer. The molecular mechanism underlying tumor development is however unknown. In the present study, we show that inhibition of NF-kappaB signaling in mouse skin by targeted expression of degradation resistant IkappaB-alpha generates transgenic keratinocytes unable to arrest the cell cycle in response to DNA damage induced by gamma-radiation. The results indicate that transgenic keratinocytes have a defect at the G1-S checkpoint whereas the G2-M checkpoint response was found to be intact. However, transgenic keratinocytes still respond by induction of the cyclin dependent kinase inhibitor p21(Cip1/Waf) after exposure to gamma-radiation. In the spontaneous skin tumors that develop in transgenic mice no mutations were found in the Ha-ras or p53 gene, suggesting that inhibition of NF-kappaB signaling in skin can induce cancer development independently of initiating mutations in the Ha-ras gene or additional mutations in the p53 gene. These findings demonstrate an involvement of NF-kappaB signaling in the DNA damage response and cell cycle checkpoint control in the skin.

Animals↗

PubMed2002

Human Krüppel-like factor5/KLF5: synergy with NF-kappaB/Rel factors and expression in human skin and hair follicles.

In this report we describe the identification of Krüppel-like factor 5 (KLF5/BTEB2) in a yeast one-hybrid screen using a keratinocyte-specific, NF-kappaB binding site as bait. The KLF5 cDNA encodes a larger protein of 457 aa rather than the earlier reported protein of 209 aa. The full-length KLF5 functions as a transactivator in HepG2 cells, and the stimulation of cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) can modulate its transcriptional activity. Overexpression of KLF5 leads to an increase in the TPA response from VLTRE, a TPA-inducible enhancer element that shows keratinocyte specificity with respect to Rel/NF-kappaB binding. The KLF5-mediated transcriptional increase is not observed in the presence of overexpressed NF-kappaB inhibitor, IkappaBalpha. Cotransfection of KLF5 and the p65 subunit of NF-kappaB, results in a synergistic transactivation of the VLTRE-luciferase reporter. The KLF5 mRNA and the protein is expressed in keratinocytes and throughout the adult human epidermis. Its expression is especially strong in the matrix and the inner root sheath cuticle layer of the hair follicle, sebaceous glands and sweat glands. Considering the TPA-responsiveness and expression pattern, we propose that KLF5 like another member of its family KLF4/GKLF may play an important role in skin morphogenesis and carcinogenesis potentially via its interaction with NF-kappaB factors.

Animals↗