Hormonal regulation and cell-specific expression of endothelin-converting enzyme 1 isoforms in bovine ovarian endothelial and steroidogenic cells

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.     

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.     

Expression of human endothelin-converting enzyme isoforms: role of angiotensin II

A key step in endothelin-1 (ET-1) synthesis is the proteolytic cleavage of big ET-1 by the endothelin-converting enzyme-1 (ECE-1). Four alternatively spliced isoforms, ECE-1a to ECE-1d, have been discovered; however, regulation of the expression of specific ECE-1 isoforms is not well understood. Therefore, we stimulated primary human umbilical vein endothelial cells (HUVECs) with angiotensin II (Ang II). Furthermore, expression of ECE-1 isoforms was determined in internal mammary arteries of patients undergoing coronary artery bypass grafting surgery. Patients had received one of 4 therapies: angiotensin-converting enzyme inhibitors (ACE-I), Ang II type 1 receptor blockers (ARB), HMG-CoA reductase inhibitors (statins), and a control group that had received neither ACE-I, ARB (that is, treatment not interfering in the renin-angiotensin system), nor statins. Under control conditions, ECE-1a is the dominant isoform in HUVECs (4.5+/-2.8 amol/microg RNA), followed by ECE-1c (2.7+/-1.0 amol/microg), ECE-1d (0.49+/-0.17 amol/microg), and ECE-1b (0.17+/-0.04 amol/microg). Stimulation with Ang II did not change the ECE-1 expression pattern or the ET-1 release. We found that ECE-1 mRNA expression was higher in patients treated with statins than in patients treated with ARB therapy (5.8+/-0.76 RU versus 3.0+/-0.4 RU), mainly attributed to ECE-1a. In addition, ECE-1a mRNA expression was higher in patients receiving ACE-I therapy than in patients receiving ARB therapy (1.68+/-0.27 RU versus 0.83+/-0.07 RU). We conclude that ECE-1a is the major ECE-1 isoform in primary human endothelial cells. Its expression in internal mammary arteries can be regulated by statin therapy and differs between patients with ACE-I and ARB therapy.     

Expression and localization of human endothelin-converting enzyme-1 isoforms in symptomatic atherosclerotic disease and saphenous vein

Endothelin-converting enzyme (ECE-1) is a critical enzyme in the production of the potent vasoconstrictor peptide endothelin (ET-1). It has previously been shown that the levels of both ET-1 and ECE-1 are raised in atherosclerosis, but the possible relevance of the isoforms of ECE-1 in these changes has not yet been investigated. The aim of this study was to examine the expression of the ECE-1a and ECE-1c isoforms in human atherosclerotic pathologies. Immunohistochemical analysis was carried out on sections from atherosclerotic and non-atherosclerotic vascular tissue using a combination of ECE-1 isoform-specific antibodies, anti-alpha-actin antibodies to identify smooth muscle cells (SMC) and anti-CD68 antibodies to identify macrophages. ECE-1 isoform expression was also examined in cultured SMC and in macrophages isolated from human blood. Results indicated differences in isoform expression in atherosclerotic lesions, with distinct patterns of staining for ECE-1a and ECE-1c. ECE-1c immunoreactivity was seen in macrophages, and also correlated with actin staining. ECE-1a was also localized to macrophages and SMC. Results of this study suggest that these local changes influence the expression patterns of the ECE-1 isoforms within individual cell types. Correlation of these isoform expression patterns with the stage of atherosclerosis could provide novel indicators of disease progression.     

Production of interleukin-1 receptor antagonist isoforms by microglia in mixed rat glial cells stimulated by lipopolysaccharide

Although the natural interleukin-1 receptor antagonist (IL-1Ra) has been shown to be produced by microglial cells in response to immune stimuli, nothing was known about the ability of these cells in primary culture to produce the different isoforms of IL-1Ra. Using RT-PCR, we first confirmed that mixed glial cell cultures from newborn rats respond to the cytokine inducer, lipopolysaccharide, by synthesizing IL-1Ra mRNA. Using double immunostaining, we showed that IL-1Ra was detected in microglia but not in astrocytes. Using Western blotting, we finally demonstrated that the IL-1Ra1 isoform was secreted in the supernatant of mixed glial cell cultures, and its production increased in response to lipopolysaccharide. The three different IL-1Ra isoforms were constitutively expressed in cell lysates and their levels increased after lipopolysaccharide treatment, except for IL-1Ra3. These results point to the ability of microglial cells in primary culture to produce the different isoforms of IL-1Ra.     

Identification of a novel alternatively spliced variant endothelin converting enzyme-1 lacking a transmembrane domain

Endothelin-converting enzyme (ECE)-1 cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (ECE-1a, 1b, 1c, and 1d) have been identified to date, they differ only in their amino terminus and share the catalytic domain located in the C-terminal end. In addition to full-length ECE-1 forms, we identified novel, alternatively spliced messenger RNAs (mRNAs) of ECE-1b, 1c, and 1d. These splice variants (SVs) lack exon 3', which codes for the transmembrane (TM) region and is present in full-length forms. SV mRNAs were highly expressed in endothelial cells (EC) derived from macrovascular and microvascular beds. Analyses of ECE-1d and its SV forms in stably transfected human embryonic kidney (HEK)-293 cells revealed that both proteins were recognized by antibodies to C-terminal ECE-1, but an antibody to the N-terminal only bound ECE-1d. The novel protein, designated ECE-1sv, has an apparent molecular weight of 75 kDa. ECE-1sv lacks the TM sequence (or signal peptide) and, therefore, is expected to remain cytosolic. Presence of ECE-1sv in different cellular compartments than the full-length forms of the ECE-1 may suggest a distinct physiologic role for these proteins.     

Phosphorylation of endothelin converting enzyme-1 isoforms: relevance to subcellular localization

Endothelin-converting enzyme (ECE)-1 is a metalloenzyme with four subisoforms, which differ only in their amino-terminal domain. ECE-1a and c are the most common isoforms and are found at the plasma membrane and in the Golgi complex, whereas ECE-1b displays lysosomal localization. We have recently shown that ECE-1a but not ECE-1b also colocalizes with nuclear membrane markers, and that maintenance of cells in high glucose (25 mM) promotes relocalization of ECE-1a from the membrane to the intracellular compartment. To investigate the mechanisms involved in this process, we conducted a search for potential phosphorylation sites, which yielded a different number of putative sites for protein kinase (PK)-C and PKA in the amino-terminal region. Stimulation of Chinese hamster ovary (CHO) cells expressing a green fluorescent protein (GFP)-tagged human ECE-1a or ECE-1b with 100 nM phorbol myristate acetate (PMA) resulted in phosphorylation of ECE-1a, as determined by immunoprecipitation with an antibody to GFP followed by immunoblotting with an antibody to phosphoserine. Stimulation of cells with PMA also promoted intracellular relocalization, as seen in cells grown under high-glucose conditions. Incubation of cells grown in 25 mM glucose with the PKC inhibitor, calphostin C (100 nM), partially prevented the relocalization of ECE-1a from the plasma membrane to intracellular compartments. Stimulation of cells with 100 nM forskolin caused phosphorylation of ECE-1b and not ECE-1a, which is consistent with the lack of a putative PKA site in the ECE-1a amino-terminal sequence. Although phosphorylation is not required for ECE-1 enzymatic activity, these results suggest that ECE-1 isoforms are phosphorylated and that phosphorylation might play an important role in the regulation of intracellular trafficking of ECE-1 subisoforms.     

Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo

Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.     

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated "rabbit TM-beta", contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of 117 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein). It differs from rabbit skeletal muscle beta-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-beta gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Cα and ‐β

Activity of protein kinase C (PKC), and in particular the PKCγ‐isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity‐dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca²⁺‐dependent PKC isoforms, PKCα and ‐β, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild‐type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCβ, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild‐type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCα‐deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild‐type cells. Together with our previous work on the role of PKCγ, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCγ‐isoform, which is highly activated by developmental processes. The PKCα isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCβ isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 95–109, 2003     

A single human myosin light chain kinase gene (MLCK; MYLK)

The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garcia et al., 1997, Am. J. Respir. Cell Mol. Biol. 16, 489-494) except for a deletion of nucleotides 1428-1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081-5233 (MLCK 3a, D3), double deletions of nucleotides 1428-1634 and 5081-5233 (MLCK 3b), and nucleotide deletions 4534-4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5' and 3' neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.     

P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and β-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms.