Endocannabinoids and endocannabinoid-like compounds modulate hypoxia-induced permeability in CaCo-2 cells via CB1, TRPV1, and PPARα.

BACKGROUND AND PURPOSE: We have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion. EXPERIMENTAL APPROACH: Caco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O2), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists. KEY RESULTS: Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB1). Basolateral application of NADA (via TRPV1), OA (via CB1 and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability. CONCLUSIONS AND IMPLICATIONS: A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB1 antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.

Genomic duplication in Dyggve Melchior Clausen syndrome, a novel mutation mechanism in an autosomal recessive disorder.

BACKGROUND: Dyggve Melchior Clausen syndrome (DMC) is a severe autosomal recessive skeletal dysplasia associated with mental retardation. Direct sequencing of genomic DNA has identified causative mutations in the gene Dymeclin (chromosome 18q12-21), with the majority predicting the generation of a truncated protein product. OBJECTIVE: To carry out molecular genetic studies in three DMC kindreds. RESULTS: Two novel nonsense mutations and two complex genomic duplication events resulting in exon repetition were identified. CONCLUSIONS: Exon dosage assessment or mRNA analysis, in addition to direct genomic DNA sequencing, should be employed in the investigation of DMC affected individuals. Genomic duplication may be the causative mutation mechanism in other autosomal recessive disorders.

Sequence, structure and pathology of the fully annotated terminal 2 Mb of the short arm of human chromosome 16.

We have sequenced 1949 kb from the terminal Giemsa light band of human chromosome 16p, enabling us to fully annotate the region extending from the telomeric repeats to the previously published tuberous sclerosis disease 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes. This region can be subdivided into two GC-rich, Alu-rich domains and one GC-rich, Alu-poor domain. The entire region is extremely gene rich, containing 100 confirmed genes and 20 predicted genes. Many of the genes encode widely expressed proteins orchestrating basic cellular processes (e.g. DNA recombination, repair, transcription, RNA processing, signal transduction, intracellular signalling and mRNA translation). Others, such as the alpha globin genes (HBA1 and HBA2), PDIP and BAIAP3, are specialized tissue-restricted genes. Some of the genes have been previously implicated in the pathophysiology of important human genetic diseases (e.g. asthma, cataracts and the ATR-16 syndrome). Others are known disease genes for alpha thalassaemia, adult polycystic kidney disease and tuberous sclerosis. There is also linkage evidence for bipolar affective disorder, epilepsy and autism in this region. Sixty-three chromosomal deletions reported here and elsewhere allow us to interpret the results of removing progressively larger numbers of genes from this well defined human telomeric region.

Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster.

We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

Characterization of a widely expressed gene (LUC7-LIKE; LUC7L) defining the centromeric boundary of the human alpha-globin domain.

We have identified the first gene lying on the centromeric side of the alpha-globin gene cluster on human 16p13.3. The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the alpha-globin genes. This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5' splice site selection. To examine the role of the 16pHQG;16 gene in delimiting the extent of the alpha-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse alpha-cluster on chromosome 11. Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human alpha-globin cluster to within an 8-kb segment of chromosome 16.

alpha-thalassemia resulting from a negative chromosomal position effect.

To date, all of the chromosomal deletions that cause alpha-thalassemia remove the structural alpha genes and/or their regulatory element (HS -40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human alpha cluster, next to a structurally normal alpha-globin gene, and silences its expression. During development, the CpG island associated with the alpha-globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the alpha-globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.

Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene.

CDP, a ubiquitous homeoprotein homologous to Drosophila cut, is implicated as a transcriptional repressor in several developmental systems. It contains four independent DNA binding domains: three "cut repeats" plus the homeodomain. The murine Cux/CDP gene spans more than 200 kb and is composed of at least 21 exons. We designed a targeting construct to replace the first cut repeat with a neomycin resistance cassette, introducing a nonsense mutation after position 1319 of the 4.5-kb reading frame of Cux/CDP. We expected to generate a truncated product of approximately 60 kDa with this construct, but instead we obtained mice expressing a mutant form of the protein, with an internal deletion of 246 amino acids encompassing cut repeat 1, but intact in the C-terminal region. Ribonuclease protection assays and direct sequencing of mutant cDNA obtained by RT-PCR demonstrate skipping of exons 10 and 11 in the mutant. Homozygous mutant mice, designated Cux/CDPDeltaCR1, display a phenotype characterized by curly vibrissae and wavy hair. We also observed a high degree of pup loss in litters born to mutant females, most likely on a nutritional basis. The mutant protein is present at levels slightly greater than wild-type, but exhibits the same tissue distribution as wild-type protein, and has approximately normal affinity for known target sequences (though no DNA targets identified to date require the first cut repeat for binding). These results support the hypothesis that the different DNA binding domains of the ubiquitous Cux/CDP protein are responsible for regulation of different genes in diverse tissues during development.

CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro.

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (>> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

Repressor activity of CCAAT displacement protein in HL-60 myeloid leukemia cells.

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.