Expression pattern of alternatively spliced PECAM-1 isoforms in hematopoietic cells and platelets

PECAM-1 (CD31) is a cell adhesion molecule that is highly expressed in the endothelium. Hematopoietic cells including platelets, monocytes, neutrophils, and some T cells also express moderate levels of PECAM-1. PECAM-1 undergoes alternative splicing generating a number of isoforms in the endothelium. However, the expression of PECAM-1 isoforms in hematopoietic cells and platelets has not been determined. Here, we examined the expression pattern of PECAM-1 isoforms in human and rodent hematopoietic cells and platelets by RT-PCR and DNA sequencing analysis. Our results showed that multiple PECAM-1 isoforms are expressed in a cell-type and species-specific pattern. We identified seven human PECAM-1 isoforms, six murine PECAM-1 isoforms, and four rat PECAM-1 isoforms. The full-length PECAM-1 was the predominant isoform detected in human cells. The PECAM-1 isoforms that lack exon 14 and 15 (delta14&15) or delta12,14&15 were the predominant isoform in rodent cells. In addition, we identified a novel PECAM-1 isoform, delta13&14, in human hematopoietic cells. Thus, hematopoietic cells express multiple isoforms of PECAM-1 in a pattern similar to that observed in the endothelium of the same species. The regulated expression of these isoforms may be important during hematopoiesis and transendothelial migration.     

Thrombospondin-1 deficient mice exhibit an altered expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature and endothelial cells

We have previously shown that thrombosponsin-1 (TSP1) and PECAM-1 are components of a regulatory switch whose reciprocal regulation in the endothelial cells (EC) promotes an angiogenic or a differentiated, quiescent phenotype. The physiological role TSP1 plays in modulation of PECAM-1 expression and function during vascular development and angiogenesis remains largely unknown. Here we demonstrate that PECAM-1 undergoes alternative splicing in its cytoplasmic domain generating eight isoforms in the retinal vasculature of wild type and TSP1-/- mice. All PECAM-1 isoforms examined contained exon 13. The frequency of PECAM-1 isoform(s) containing exon 14 was significantly higher during early stages of retinal vascularization, which decreased during later stages of retinal vascularization in wild type mice. In contrast, the frequency of exon 14 containing PECAM-1 isoform(s) did not significantly change during retinal vascularization in TSP1-/- mice. They consistently expressed higher number of isoforms with exon 14 during later stages of retinal vascularization. The higher level of PECAM-1 isoforms with exon 14 was also observed in cultured TSP1-/- retinal EC compared to wild type retinal EC. This was consistent with increased amounts of Src and SHP-2 associated with PECAM-1, and enhanced migration and proliferation in TSP1-/- retinal EC. These data suggest PECAM-1 signaling in the endothelium is modulated by its alternative splicing during retinal vascular development and angiogenesis, which may be impacted by TSP1 expression.     

Kinetic expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) during embryonic stem cell differentiation

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as a marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. However, the expression of PECAM-1 isoforms in ES cells has not been determined. The present study was designed to determine the role of PECAM-1 isoforms during in vitro endothelial differentiation of ES cells. It was found that undifferentiated ES cells expressed high level of PECAM-1, which primarily located at cell-cell junction, but the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. In addition, undifferentiated ES cells were found the expressed all eight known alternatively spliced PECAM-1 isoforms, among them the expression of PECAM-1 isoforms lacking exon 15 or 14&15 was predominant. Quantitative analysis revealed a significant increase in the expression of PECAM-1 isoform lacking exon 12&14&15 as vascular development of ES cells. These results indicate a constitutive expression of PECAM-1 in undifferentiated murine ES cells and suggest a developmental role of PECAM-1 isoform changes during vasculogenesis and angiogenesis.     

PECAM-1 isoform-specific regulation of kidney endothelial cell migration and capillary morphogenesis

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in angiogenesis through its involvement in endothelial cell-cell and cell-matrix interactions and signal transduction. Recent studies indicate that the cytoplasmic domain of PECAM-1 plays an important role in its cell adhesive and signaling properties. However, the role PECAM-1 isoforms play during angiogenic events such as cell adhesion and migration requires further delineation. To gain insight into the role PECAM-1 plays during vascular development and angiogenesis, we examined the expression pattern of PECAM-1 isoforms during kidney vascularization. We show that multiple isoforms of PECAM-1 are expressed during renal vascular development with different frequencies. The PECAM-1 that lacks exons 14 and 15 (14&15) was the predominant isoform detected in the renal vasculature. To further study PECAM-1 isoform-specific functions we isolated kidney endothelial cells (EC) from wild-type and PECAM-1-deficient (PECAM-1–/–) mice with B₄-lectin-coated magnetic beads. PECAM-1–/– kidney EC showed reduced migration, inability to undergo capillary morphogenesis in Matrigel, dense peripheral focal adhesions, and peripheral cortical actin distribution compared with wild-type cells. PECAM-1–/– kidney EC secreted increased amounts of fibronectin and decreased amounts of tenascin-C and thrombospondin-1. Reexpression of 14&15, but not full-length, PECAM-1 in PECAM-1–/– kidney EC restored cell migration and capillary morphogenesis defects. Thus PECAM-1 may regulate the adhesive and migratory properties of kidney EC in an isoform-specific fashion through modulation of integrin activity and extracellular matrix protein expression. Our results indicate that regulated expression of specific PECAM-1 isoforms may enable EC to accommodate the different stages of angiogenesis. CD31; alternative splicing; angiogenesis; integrins; extracellular matrix     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.     

Tyrosine Residue in Exon 14 of the Cytoplasmic Domain of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) Regulates Ligand Binding Specificity

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19– amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell–cell aggregation while those variants missing exon 14 mediate homophilic cell–cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5–amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.     

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.     

Cyclic AMP-dependent protein kinase A regulates the alternative splicing of CaMKIIδ

Ca(2+)/calmodulin-dependent protein kinase (CaMK) IIδ is predominantly expressed in the heart. There are three isoforms of CaMKIIδ resulting from the alternative splicing of exons 14, 15, and 16 of its pre-mRNA, which is regulated by the splicing factor SF2/ASF. Inclusion of exons 15 and 16 or of exon 14 generates δA or δB isoform. The exclusion of all three exons gives rise to δC isoform, which is selectively increased in pressure-overload-induced hypertrophy. Overexpression of either δB or δC induces hypertrophy and heart failure, suggesting their specific role in the pathogenesis of hypertrophy and heart failure. It is well known that the β-adrenergic-cyclic AMP-dependent protein kinase A (PKA) pathway is implicated in heart failure. To determine the role of PKA in the alternative splicing of CaMKIIδ, we constructed mini-CaMKIIδ genes and used these genes to investigate the regulation of the alternative splicing of CaMKIIδ by PKA in cultured cells. We found that PKA promoted the exclusion of exons 14, 15, and 16 of CaMKIIδ, resulting in an increase in δC isoform. PKA interacted with and phosphorylated SF2/ASF, and enhanced SF2/ASF's activity to promote the exclusion of exons 14, 15, and 16 of CaMKIIδ, leading to a further increase in the expression of δC isoform. These findings suggest that abnormality in β-adrenergic-PKA signaling may contribute to cardiomyopathy and heart failure through dysregulation in the alternative splicing of CaMKIIδ exons 14, 15, and 16 and up-regulation of CaMKIIδC.     

Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA-sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full-length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin-binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full-length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full-length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected generating predominantly muscle-specific Tpm1 isoforms in adult hearts. We found that the RNA-binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA-binding protein PTBP1. In sum, we defined full-length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA-binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle-enriched genes.     

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.     

Identification and characterization of two new human mu opioid receptor splice variants,hMOR-1O and hMOR-1X

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene (Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1O and hMOR-1X. The full-length cDNAs of hMOR-1O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.     

Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.     

<Emphasis Type="SmallCaps">l</Emphasis>-Dopa decarboxylase expression profile in human cancer cells

l-Dopa decarboxylase (DDC) catalyses the decarboxylation of l-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5′-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5′UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.