Tissue-specific expression of Cre recombinase from the Tgfb3 locus

Tgfb3, a member of the TGF-beta superfamily, is tightly regulated, both spatially and temporally, during embryogenesis. Previous mouse knockout studies have demonstrated that Tgfb3 is absolutely required for normal palatal fusion and pulmonary development. We have generated a novel tool to ablate genes in Tgfb3-expressing cells by targeting the promoterless Cre-pgk-Neo cassette into exon 1 of the mouse Tgfb3 gene, which generates a functionally null Tgfb3 allele. Using the Rosa26 reporter assay, we demonstrate that Cre-induced recombination was already induced at embryonal day 10 (E10) in the ventricular myocardium, limb buds, and otic vesicles. At E14, robust recombination was detected in the prefusion palatal epithelium. Deletion of the TGF-beta type I receptor Alk5 (Tgfbr1) specifically in Tgfb3 expressing cells using the Tgfb3-Cre driver line lead to a cleft palate phenotype similar to that seen in conventional Tgfb3 null mutants. In addition, Alk5/ Tgfb3-Cre mice displayed hydrocephalus, and severe intracranial bleeding due to germinal matrix hemorrhage.     

p38 Mitogen-Activated Protein Kinase Functionally Contributes to Chondrogenesis Induced by Growth/Differentiation Factor-5 in ATDC5 Cells

Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta (TGF-beta) superfamily have focused on Smad proteins, but have paid little attention to mitogen-activated protein (MAP) kinase cascades. Here we demonstrate that growth/differentiation factor-5 (GDF-5), but neither bone morphogenetic protein-2 (BMP-2) nor TGF-beta1, fully promotes the early phase of the chondrogenic response by inducing cellular condensation followed by cartilage nodule formation in a mouse chondrogenic cell line, ATDC5. We investigated which, if any, of the three major types of MAP kinase plays a functional role in the promotion of chondrogenesis induced by GDF-5. GDF-5 induced phosphorylation of p38 MAP kinase and extracellular signal-regulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK). The phosphorylation of p38 MAP kinase was also induced by BMP-2 and TGF-beta1. An inhibitor of p38 and p38 beta MAP kinase, SB202190, showed complete inhibition of cartilage nodule formation but failed to affect alkaline phosphatase (ALP) activity induced by GDF-5. Expression of the type II collagen gene, a hallmark of chondrogenesis in vertebrates, was also induced by GDF-5 treatment and strongly suppressed by SB202190. On the other hand, although an inhibitor of MAP/ERK kinase, PD98059, inhibited the rapid phosphorylation of ERK by GDF-5, it inhibited neither ALP activity nor cartilage nodule formation induced by GDF-5. These results strongly suggest that the p38 MAP kinase cascade is involved in GDF-5 signaling pathways and that a role of the p38 MAP kinase pathway is necessary over a longer period to promote chondrogenesis in ATDC5 cells.     

The cell adhesion molecule CEACAM1-L is a substrate of caspase-3-mediated cleavage in apoptotic mouse intestinal cells

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.     

Characterization and identification of the inhibitory domain of GDF-8 propeptide

GDF-8 is a negative regulator of skeletal muscle mass. The mechanisms which regulate the biological activity of GDF-8 have not yet been elucidated. Analogous to the TGF-beta system, GDF-8 propeptide binds to and inhibits the activity of GDF-8. In these studies, we define the critical domain of the GDF-8 propeptide necessary for inhibitory activity. Two molecules of GDF-8 propeptide monomer inhibit the biological activity of one molecule of GDF-8 homodimer. Although the propeptide contains N-linked glycosylation when synthesized in mammalian cells, this glycosylation is not necessary for the inhibition of GDF-8. Taking advantage of the bacterial expression system, we express and purify GDF-8 propeptide which retains full inhibitory activity. To define the functional regions of the propeptide, we express a series of truncated GST-propeptide fusion proteins and examined their inhibitory activity. We observe that fusion proteins containing the C-terminal region (amino acid residues 99-266) are very stable, but do not exhibit inhibitory activity; while fusion proteins containing the N-terminal region (amino acid residues 42-115) are labile but contain essential inhibitory activity. The data suggest that the C-terminal region may play a role in the stability of the GDF-8 propeptide and that the inhibitory domain is located in the region between amino acids 42 and 115.     

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.     

A nitrite transporter associated with nitrite uptake by higher plant chloroplasts

Chloroplasts take up cytosolic nitrite during nitrate assimilation. In this study we identified a nitrite transporter located in the chloroplasts of higher plants. The transporter, CsNitr1-L, a member of the proton-dependent oligopeptide transporter (POT) family, was detected during light-induced chloroplast development in de-etiolating cucumber seedlings. We detected a CsNitr1-L-green fluorescent protein (GFP) fusion protein in the chloroplasts of leaf cells and found that an immunoreactive 51 kDa protein was present in the isolated inner envelope membrane of chloroplasts. CsNitr1-L has an isoform, CsNitr1-S, with an identical 484 amino acid core sequence; however, in CsNitr1-S the 120 amino acid N-terminal extension is missing. Saccharomyces cerevisiae cells expressing CsNitr1-S absorbed nitrite from an acidic medium at a slower rate than mock-transformed control cells, and accumulated nitrite to only one-sixth the concentration of the control cells, suggesting that CsNitr1-S enhances the efflux of nitrite from the cell. Insertion of T-DNA in a single CsNitr1-L homolog (At1g68570) in Arabidopsis resulted in nitrite accumulation in leaves to more than five times the concentration found in the wild type. These results show that it is possible that both CsNitr1-L and CsNitr1-S encode efflux-type nitrite transporters, but with different subcellular localizations. CsNitr1-L may possibly load cytosolic nitrite into chloroplast stroma in the chloroplast envelope during nitrate assimilation. The presence of genes homologous to CsNitr1-L in the genomes of Arabidopsis and rice indicates that facilitated nitrite transport is of general physiological importance in plant nutrition.     

Four fibroblast tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene via alternative RNA splicing and the use of two promoters

cDNA clones encoding four rat tropomyosin isoforms, termed TM-2, TM-3, TM-5a, and TM-5b, were isolated and characterized. All are derived from the alpha-tropomyosin gene via alternative RNA processing and the use of two alternate promoters. The cDNA sequences predict that TM-2 and TM-3 both contain 284 amino acids and differ from each other only at an internal region of the protein from amino acids 189 through 213, due to alternative splicing of exons 6a and 6b. TM-5a and TM-5b both contain 248 amino acids and differ from each other only at an internal exon encoding amino acids 153 through 177, also due to alternative splicing of exons 6a and 6b. The differences in the amino acid sequence encoded by these alternate exons affects the theoretical actin-binding pattern of the tropomyosins, such that TM-5b is expected to bind actin with greater affinity than TM-5a. TM-2 and TM-3 are transcribed from the upstream promoter, and TM-5a and TM-5b are transcribed from an internal promoter. In addition, all four isoforms contain the identical COOH-terminal coding region. RNA protection analyses revealed that the mRNA for each isoform is expressed in a number of different tissues and cell types, although the expression of some isoforms is restricted to particular cell types. Furthermore, the expression of mRNA encoding these isoforms was found to be altered in a number of different virally transformed cell lines. The changes in the expression of tropomyosin mRNAs in transformed cells reflect changes in the relative use of the two promoters, as well as the relative use of alternatively spliced exons 6a and 6b.     

Isolation and mapping of a human lung-specific gene, TSA1902, encoding a novel chitinase family member.

Using differential display technique, we have isolated a novel human gene expressed specifically in the lung. Two forms of the gene, designated TSA1902, were transcribed by alternate mRNA splicing. The transcribed mRNAs, termed TSA1902-L and TSA1902-S, putatively encode proteins of 368 and 315 amino acids, respectively, which show high similarity to human chitotriosidase protein. The N-terminal region of TSA1902-L protein contains the conserved active site residues (DXXDXDXE) of the catalytic center of various chitinases which are essential for chitinase activity. The deduced protein sequence of TSA1902-S, however, does not possess this active site, with the N-terminal 54 amino acids present in TSA1902-L protein having been deleted. Both proteins lacked the secretory sequence of N-termini and, judging from the hydropathy profile, may be soluble proteins in the cytoplasm. Chromosomal mapping by radiation hybrid analysis localized this gene to the chromosome 1p13.1-p21.3.     

cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. To identify novel genes whose protein products may mediate cellular responses to this factor, a cDNA library was made from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated for 3 days with TGF-beta. The library was screened by differential hybridization and a cDNA clone, beta ig-h3, was isolated. This gene was induced up to 20-fold in A549 cells after 2 days of treatment with TGF-beta 1. It was also induced in several other cell lines, including PC-3 and H2981. DNA sequence analysis of beta ig-h3 indicated that it encoded a novel protein, beta IG-H3, of 683 amino acids, which contained an amino-terminal secretory sequence and a carboxy-terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand recognition site for several integrins. beta IG-H3 also contained short amino acid regions homologous to similar regions in Drosophila fasciclin-I and four homologous internal domains, which can be folded into a potential bivalent structure and could act as a bridge between cells expressing the appropriate ligand. beta ig-h3 RNA was detected in several cell lines and tissues. COS cells transfected with plasmids encoding beta IG-H3 secreted a major 68-kD protein that was detected by immunoblotting using antipeptide antibodies. Since beta ig-h3 is induced in several cell lines whose proliferation is affected by TGF-beta 1, it may be involved in mediating some of the signals of this multifunctional growth modulator.     

cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed.     

Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

TGF-beta type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-beta superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-beta type II receptor appears to bind only TGF-beta, whereas TGF-beta type I receptor (ALK5) also binds to ligands in addition to TGF-beta. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-beta type II receptor (Wnt1-Cre;Tgfbr2(fl/fl)) and TGF-beta type I receptor/Alk5 (Wnt1-Cre;Alk5(fl)(/fl)) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Expression of SLC2A9 Isoforms in the Kidney and Their Localization in Polarized Epithelial Cells

Background

Many genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro.

Methodology/Principal Findings

Isoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type.

Conclusions/Significance

SLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking.