TIMAP, a novel CAAX box protein regulated by TGF-beta 1 and expressed in endothelial cells

Wangsen Cao1, Subhendra N. Mattagajasingh2, Hangxue Xu1, Kwanghee Kim2, Wolfgang Fierlbeck2, Jie Deng1, Charles J. Lowenstein1, and Barbara J. Ballermann2

1 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and 2 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
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ABSTRACT
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Representational difference analysis of the glomerular endothelial cell response to transforming growth factor-beta 1 (TGF-beta 1) revealed a novel gene, TIMAP (TGF-beta -inhibited membrane-associated protein), which contains 10 exons and maps to human chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed in all cultured endothelial and hematopoietic cells. The frequency of the TIMAP SAGE tag is much greater in endothelial cell SAGE databases than in nonendothelial cells. Immunofluorescence studies of rat tissues show that anti-TIMAP antibodies localize to vascular endothelium. TGF-beta 1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process. The TIMAP protein contains five ankyrin repeats, a protein phosphatase-1 (PP1)-interacting domain, a COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3, and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein localized to the plasma membrane in a CAAX box-dependent fashion. Hence, TIMAP is a novel gene highly expressed in endothelial and hematopoietic cells and regulated by TGF-beta 1. On the basis of its domain structure, TIMAP may serve a signaling function, potentially through interaction with PP1.

hematopoietic cells; rat; representational difference analysis; glomerular endothelial cells; human; vascular endothelial cells


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MEMBERS OF THE transforming growth factor-beta (TGF-beta ) family of homodimeric extracellular ligands, which include TGF-beta s, activins, and bone morphogenetic proteins, regulate many cellular functions related to development and differentiation (23, 24).

Cell signaling by the ligands of the TGF-beta superfamily occurs through activation of cell surface receptor serine/threonine kinases and subsequent activation of intracellular signaling molecules, the Smads (6, 23). TGF-beta 1, the most studied member of the mammalian TGF-beta s, binds directly to type II TGF-beta receptor, a constitutively active receptor serine/threonine kinase. This interaction also requires an accessory cell surface proteoglycan, either beta -glycan (20) or endoglin (4), the latter expressed predominantly in endothelial cells. On TGF-beta 1 binding, recruitment of type I TGF-beta receptors into a heteromeric complex with type II receptors results in phosphorylation of type I receptors and activation of their kinase activity. Transient association of receptor-specific Smad2 and/or Smad3 (R-Smads) with active type I receptors causes R-Smad phosphorylation, which is followed by the formation of a heteromeric complex between R-Smad and the related protein Smad4 (Co-Smad). The complex then moves to the nucleus, where it regulates transcription by interacting with cooperating transcriptional factors and Smad-responsive promoter elements. It is now understood that the R-Smad-Co-Smad complex can interact with diverse transcriptional regulators, for instance, transcriptional factors interacting with activator protein-1 sites (18, 43), transcriptional cofactors, such as FAST (44), and transcriptional repressors, such as Ski, SnoN, and TGIF (34, 40, 41).

Members of the TGF-beta superfamily are critical in controlling cell proliferation, matrix synthesis, adhesion to matrix, and apoptosis, and absence of some components of the TGF-beta pathway promotes tumor formation (22, 23, 37). The specific effect of TGF-beta 1 and its family members is highly cell dependent (24). In cultured endothelial cells, TGF-beta 1 inhibits cell proliferation (36), alters endothelial cell-matrix interactions (28), and induces microvascular endothelial cell apoptosis (5), an effect that can be rescued by activation of integrin-beta 1 (29). In mice, deficiency of type II TGF-beta receptor (27) or endoglin, the class III TGF-beta coreceptor expressed by endothelial cells (17), results in an embryonic lethal phenotype with aberrant vascular morphogenesis in the yolk sac. Deficiency of ALK-1, a type I TGF-beta receptor, also lethal to embryos, has a very similar phenotype (26). The findings in humans that mutations in endoglin (25) and ALK-1 (11) result in structural abnormalities of the vasculature with the phenotype of hereditary hemorrhagic telangiectasia also suggest that TGF-beta signaling processes are critical in the development and maintenance of normal vascular structures.

We previously reported that TGF-beta plays a critical role in glomerular capillary morphogenesis. TGF-beta 1 stimulates assembly of cultured glomerular endothelial (GEN) cells into capillaries, with the remainder of the cells undergoing apoptosis (5). Capillary formation by GEN cells in vitro is abrogated in cells expressing the dominant-negative type II TGF-beta receptor and also by neutralizing TGF-beta 1 antibody. In vivo, normal glomerular capillary formation and differentiation during renal development in rats are also dependent on TGF-beta 1 (19).

Although there is ample evidence that TGF-beta 1 is involved in the formation and maintenance of blood vessel architecture, the molecules downstream from the TGF-beta 1 signaling cascade in endothelial cells are only partially understood. We therefore sought to identify previously unknown targets of the TGF-beta signaling cascade in endothelial cells. Here we report the characterization of a novel gene, TIMAP (TGF-beta -inhibited membrane-associated protein), highly expressed in endothelial cells, which is strongly repressed by TGF-beta 1. The protein product of this transcript contains several ankyrin repeats in its NH2-terminal half and a COOH-terminal CAAX box, which mediates its plasma membrane association.


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Cell lines, culture, and cell treatment. Primary bovine GEN and bovine aortic endothelial (BAE) cells were prepared as described previously (1, 2). U-937 [histiocytic lymphoma, macrophage-like cell line; CRL-1593.2, American Type Culture Collection (ATCC)], human erythrocytic leukemia (HEL; TIB-180, ATCC), lymphoblastic T cell leukemia (MOLT4; CRL-1582, ATCC), human embryonic kidney (HEK-293; CRL-1573, ATCC), human erythroleukemia (TF-1; CRL-2003, ATCC), and Dami (a human megakaryocytic cell line; obtained from Dr. Paul F. Bray, Baylor University School of Medicine, Baylor, TX) cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). Granulocyte/macrophage colony-stimulating factor (1 ng/ml) was added to TF-1 cell medium. KG-1a (human acute myelogenous leukemia, myeloblast; CCL-246.1, ATCC) and K-562 (human chronic myelogenous leukemia, lymphoblast; CCL-243, ATCC) cells were cultured in Iscove's modified Dulbecco's medium with 10% FBS. WM793 (a human melanoma cell line; obtained from Dr. Paul F. Bray), COS-7 (African green monkey kidney cell; CRL-1651, ATCC), and Madin-Darby canine kidney (MDCK; CCL-34, ATCC) cells were cultured in DMEM with 10% FBS. HeLa cells (human cervical carcinoma epithelial cell line; CCL-2, ATCC) were cultured in Eagle's minimum essential medium with 10% FBS. HCT116 cells (human colorectal carcinoma epithelial cell line; CCL-247, ATCC) were cultured in McCoy medium with 10% FBS. Human aorta endothelial cells, human umbilical vein endothelial cells, and human microvascular endothelial cells (HMEC-1) were purchased from Clonetics (San Diego, CA) and cultured in appropriate medium (Clontech) supplemented with 10% FBS, 10 µg/ml hydrocortisone (Sigma, St. Louis, MO), and 10 ng/ml epidermal growth factor (Collaborative Biomedical Products-Becton Dickinson, Bedford, MA).

In experiments delineating the TGF-beta 1 response, cells were treated with TGF-beta 1 (Becton Dickinson) at 5 ng/ml for 6 h, unless stated otherwise. Cycloheximide (CHX; Sigma) was added to some cells at 10 nM for 30 min before the TGF-beta 1 treatment. Actinomycin D (10 µg/ml) was added simultaneously with TGF-beta 1. Trichostatin A (TSA; Sigma) was added to some cells at 30 ng/ml for 30 min before TGF-beta 1 treatment.

Representational difference analysis. For representational difference analysis (RDA), cDNA was synthesized using poly(A)+ RNA template from GEN cells treated with TGF-beta 1 (GEN/TGF-beta 1 positive) or left untreated (GEN/TGF-beta 1 negative). The cDNA pools representing each cell population were digested with DpnII, ligated to an oligonucleotide linker with a 5' overhang [5'-AGCACTCTCCAGCCTCTCACCG CA-3' (R-24) and 5'-GATC TGCGGTGA-3' (R-24); complementary region for the pair is underlined], and amplified with R-24 primer to generate GEN/TGF-beta 1-positive and GEN/TGF-beta 1-negative amplicon pools. The linkers were then removed from the amplicon cDNAs by DpnII digestion and purification with Amicon 100 columns, generating "driver" cDNAs. "Tester" cDNA representing each cell population was generated by ligation of a distinct oligonucleotide linker with a 5' overhang [J-linker: 5'-ACCGACGTCGAC TATCCATGAACA-3' (J-24) and 5'-GATCTGTTCATG-3' (J-12)] to a portion of each driver pool.

Subtractive amplification was performed as previously reported (21). Briefly, tester cDNA from one cell pool was hybridized to excess driver cDNA from the second cell pool and then amplified with linker-specific primers, the linkers being present only in the tester cDNA. Sequences in the tester pool for which complementary cDNA exists in the driver pool will, on hybridization, leave one strand without linker and are not amplified. In the first round of RDA, a tester-to-driver ratio of 1:100 was used. In the second round, the J-linker on the cDNA product derived from the first round of amplification was replaced with a third oligonucleotide linker with a 5' overhang [N-linker: 5'-AGGCAACTGTGCTA TCCGAGGGAA-3' (N-24) and 5'-GATCTTCCCTCG-3'(N-12)] and then hybridized with excess driver at a ratio of 1:800. A third round of subtractive amplification, after digestion of the N-linker on the RDA product pool and replacement with J-linker, did not alter the appearance of product bands or background. RDA was performed using tester and driver cDNA from each cell population reciprocally to find TGF-beta 1-upregulated and -downregulated transcripts (Fig. 1A).


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Fig. 1.   Identification and regulation of transforming growth factor-beta (TGF-beta )-inhibited membrane-associated protein (TIMAP) mRNA. A: representational difference analysis (RDA) of glomerular endothelial (GEN) cells. GEN cells were treated with TGF-beta 1 (+) or medium alone (-). RNA was converted to cDNA amplicon pools (Amp) and then differentially amplified by PCR in an initial round (R1) and a second round (R2). up-arrow , Products obtained by subtracting GEN/TGF-beta 1-negative from GEN/TGF-beta 1-positive cells; down-arrow , products obtained by subtracting GEN/TGF-beta 1-positive from GEN/TGF-beta 1-negative cells (down-arrow ). MW, molecular weight size markers. B: Northern blot analysis of TGF-beta 1-treated GEN cells using difference products generated from the second round of RDA as probes. Ribosomal protein L19 (rpL19), not affected by TGF-beta 1, was used as loading control. Lane 1, plasminogen activator inhibitor-1 (PAI-1); lane 2, integrin-alpha 5; lane 3, TIMAP; lane 4, fibronectin; lanes 5 and 7, unknown TGF-beta 1-upregulated mRNA; lane 6, unknown TGF-beta 1-downregulated mRNA. C: TGF-beta 1 represses TIMAP in GEN and bovine aorta endothelial (BAE) cells. GEN and BAE cells were treated with or without TGF-beta 1 for 6 h and analyzed by Northern blot with TIMAP cDNA as a probe. D: time course of TIMAP downregulation by TGF-beta 1. Total RNA from GEN/TGF-beta 1-negative or GEN/TGF-beta 1-positive cells was collected at various times and hybridized with probes for TIMAP, PAI-1, and ribosomal protein L27a (rpL27a).

RDA-selected difference products were then 1) digested with DpnII, run on a 1% agarose gel, excised as individual bands, and subcloned into BamHI-digested pBluescript KS vector or 2) subcloned directly into the pCR2.1TOPO TA cloning vector (Invitrogen, San Diego, CA). Subcloned difference products were labeled with 32P and hybridized to cDNA blots of GEN/TGF-beta 1-positive and GEN/TGF-beta 1-negative amplicon pools. Those cDNAs for which differential representation in the amplicon pools was verified were sequenced using the dideoxy chain termination method. One of them, TIMAP, represents a transcript highly expressed in GEN cells in the basal state, which was strongly repressed by TGF-beta 1.

Full-length TIMAP cDNAs. The initial 278-bp fragment of bovine TIMAP (bTIMAP) cDNA generated by RDA was extended by 0.9 kb in the 3' direction using 3'-rapid amplification of cDNA ends (RACE) with mRNA from GEN cells using a Marathon 5'/3'-RACE kit (Invitrogen) and oligo 5'-CCAGATGCCCAGCTC CTGGTTAGA-3' as the 5' primer. The 1.1-kb 3'-RACE product was subcloned into the pCR2.1 TA cloning vector and sequenced, and a BAE cell lambda ZAPII cDNA library (936705, Stratagene, La Jolla, CA) was then screened by using the 1.1-kb TIMAP cDNA fragment as a probe. From 106 plaque-forming units, two clones were isolated that extended the sequence 4.3 kb in the 5' direction. A 693-bp fragment from the 5' end of one clone was then used to screen the library for a second time. Several overlapping clones that extended the sequence another ~1 kb in the 5' direction were found. All the clones were sequenced with multiple internal primers to generate the full-length bTIMAP cDNA sequence.

The bTIMAP has 80% homology to the human KIAA0823 cDNA (accession no. AB020630, GenBank), which is 5,597 bp long and lacks an initial methionine. Searches using the dbest algorithm revealed that several human expressed sequence tags (ESTs; e.g., accession nos. AI732267 and AA885365, GenBank) are derived from the same transcript as KIAA0823 and extend the sequence 259 bp upstream of the KIAA0823 5' end. The 5' end of the human TIMAP (hTIMAP) cDNA was generated by 5'-RACE using the 5'/3'-RACE kit (Invitrogen). For 5'-RACE, first-strand cDNA was generated from human aorta endothelial cell mRNA using a primer specific for the 5' end of the KIAA0823 (nt 11-36; accession no. AB020630, GenBank). A linker was added to the 5' end of the resulting cDNA, and then nested PCR was carried out with an EST-specific primer representing nt 45-74 of the human EST AA885365 (nt 127-156 of the human EST; accession no. AI732267, GenBank) and a primer against the 5' linker. The 5'-RACE product of 400 bp was subcloned into a TA cloning vector and sequenced.

Northern blots. Total RNA was harvested from various cells and from rat tissues using TRIzol (GIBCO Life Technology, Rockville, MD) and then subjected to extraction with phenol-chloroform. RNA (10-20 µg) was fractionated on a 1% formaldehyde agarose gel and transferred to a nylon membrane. 32P-labeled probes were made from various cDNA inserts (Rediprime kit, Amersham, Piscataway, NJ) and purified using a G-25 column (Amersham). The blots were hybridized at 62°C overnight in PreHyb buffer (Amersham) containing the different probes at 106 cpm/ml buffer. The Northern blots were visualized by exposure of the blot to a Kodak film or use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To control for loading, blots were reprobed with ribosomal protein L19 or L27a cDNA or with glyceraldehyde-3-phosphate dehydrogenase cDNA.

cDNA constructs and probes. An hTIMAP cDNA encoding the predicted open reading frame (ORF) was generated by RT-PCR from human aorta endothelial cell mRNA using the 5' primer 5'-GGAATTCGATGGCCAGTCACGTGGA CCT-3' and the 3' primer 5'-CGGTCGACTAGGAGATACGGCAACAGCCATG-3', in which BamHI and SalI sites are underlined. TIMAP(C564S) was constructed in the same way, except the 3' primer had a single nucleotide replacement, 5'-CGGTCGACTAGGAGATACGGGAACAGCCATG-3', which produced a serine instead of a cysteine in the CAAX box. The PCR products were digested with BamHI and SalI and ligated into pEGFP(C1) vector in BamHI/XhoI sites. The cDNA products were sequenced fully. The fusion proteins encoded by these cDNAs express enhanced green fluorescent protein (EGFP) at the NH2 terminus of TIMAP or TIMAP(C564S). The RDA products for plasminogen activator inhibitor-1 (PAI-1), ribosomal protein L19, ribosomal protein L27a, fibronectin, integrin-alpha , and laminin-beta 1 cloned into pBluescript were used as probes.

Subcellular localization. GEN, MDCK, and COS-7 cells were plated on glass coverslips coated with type I collagen on the day before transfection. On the next day, transfection with pEGFP, pEGFP/TIMAP, or pEGFP/TIMAP(C654S) plasmids was carried out with LipofectAMINE 2000 for MDCK and COS-7 cells and by Lipofectin (GIBCO BRL) for GEN cells. After 24 h, the transfected cells were observed with an Aus Jena microscope using epifluorescence. Photomicrographs were taken with an Olympus camera with Kodak Elite 400 slide film. For confocal microscopy, the coverslips were mounted in mounting buffer (100 mM n-propyl gallate, 50% glycerol in PBS, pH 7.4), and the samples were viewed with a Nikon microphot-FXA or a laser scanning confocal system (model MRC 600, Bio-Rad Laboratories) coupled to a Zeiss Axiophot microscope through a ×100 oil-immersion objective. Images were processed using Photoshop software (Adobe Systems).

Immunhistochemistry and immunofluorescence studies. Polyclonal antibodies were prepared by Zymed Laboratories (South San Francisco, CA) by immunizing rabbits with peptides representing aa 383-401 and aa 511-532 of hTIMAP. These peptides are conserved between hTIMAP and bTIMAP and are not found in MYPT3, the closest relative of TIMAP (see Fig. 3A) or in other known or predicted protein sequences. The immunoglobulins were purified using peptide affinity columns prepared with the Sulfolink kit (Pierce, Rockford, IL). Each polyclonal anti-TIMAP IgG recognized full-length, but not NH2-terminal (aa 1-90), TIMAP expressed in bacteria (not shown). In endothelial cell and rat renal glomerular lysates, both antibodies recognize two predominant bands at 67 kDa (expected molecular mass for full-length TIMAP) and at 120 kDa (not shown). The identity of the second band is not known. It may represent a posttranslationally modified TIMAP isoform or an as yet unknown sequence in which both epitopes chosen for immunization are conserved.

TIMAP expression was examined in neonatal and adult rat tissues previously fixed in 10% buffered formalin (pH 7.4) and embedded in paraffin (19). Sections (5 µm) were prepared, deparaffinized with xylene, and rehydrated in a descending series of ethanols. Endogenous peroxidases were quenched twice with 0.3% hydrogen peroxide for 10 min at room temperature. The sections were then treated with 0.4% pepsin in 0.01 N HCl at 37°C for 30 min, washed twice with PBS, and blocked with 0.5% nonfat dry milk and 5% goat serum for 30 min at room temperature. The sections were incubated in the same solution with anti-TIMAP antibodies (1:100) at 4°C overnight, allowed to equilibrate to room temperature for 20 min, and washed twice with PBS. Sections were incubated with biotinylated goat anti-rabbit antibodies (Vectastain Elite ABC kit) for 30 min at 37°C and then with streptavidin-horseradish peroxidase (HRP; Vectastain Elite ABC kit) for 10 min at 37°C and visualized with diaminobenzidine.

For dual-label immunofluorescence, the reaction was performed as described above, except HRP was visualized with the Cy3 tyramide (NEL704, New England Nuclear Life Science Products, Boston, MA). The HRP was then quenched, as described above, and the sections were incubated in the dark with mouse monoclonal anti-smooth muscle actin antibody (1:400; clone 1A4, Sigma) overnight at 4°C and then with goat anti-mouse HRP (1:1,000; New England Nuclear, Boston, MA). Visualization of the mouse monoclonal reaction product was achieved with tyramide fluorescein amplification (NEL701A, New England Nuclear). Immunofluorescence studies reflect data from five separate experiments using tissues from five different animals.

Nucleotide sequence accession number. The nucleotide sequence accession numbers in the GenBank database are AF362910 and AF362909 for hTIMAP and bTIMAP cDNA, respectively.


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Identification of TIMAP, a novel TGF-beta 1-responsive gene, in endothelial cells. To identify TGF-beta 1-responsive genes in endothelial cells, GEN cells were cultured in the presence or absence of TGF-beta 1 (5 ng/ml) for 6 h, and mRNA was isolated and analyzed by RDA (Fig. 1A). TGF-beta 1-upregulated and -downregulated RDA products were purified, subcloned, and sequenced.

Among the RDA products upregulated by TGF-beta 1 were PAI-1 (30), integrin-alpha 5 (7), fibronectin (10, 39), and laminin-beta 1 (39), all known to be strongly induced by TGF-beta 1 (Fig. 1B). Because TGF-beta 1 is known to stimulate expression of these mRNAs, these findings confirm that the RDA procedure appropriately selected TGF-beta 1-stimulated difference products. Several novel sequences were found to be induced (TGF-beta 1-upregulated genes) and others repressed (TGF-beta 1-downregulated genes) by TGF-beta 1. All difference products identified by RDA were also differentially represented in the GEN/TGF-beta 1-positive vs. the GEN/TGF-beta 1-negative amplicons (not shown). Northern blot analysis of RNA from separate experiments was then performed and confirmed that TGF-beta 1 increased expression of PAI-1, integrin-alpha 5, fibronectin, and TGF-beta 1-upregulated genes and decreased expression of TGF-beta 1-downregulated genes in GEN cells (Fig. 1B). Because the TIMAP clone was found to have partial homology to KIAA0823 (35) (accession no. AB020630, GenBank), a sequence that predicted two important functional domains, namely, a CAAX box and several ankyrin repeats, it was further characterized.

TIMAP mRNA was readily detected in bovine GEN and BAE cells (Fig. 1C) and was strongly repressed by TGF-beta 1. TIMAP expression was also observed in human aorta, umbilical vein, and microvascular endothelial cells (not shown). Hence, TIMAP is expressed in cultured large vessel and cultured microvascular endothelial cells.

TIMAP mRNA levels declined in GEN cells within 2 h and fell to nearly undetectable levels 4-24 h after TGF-beta 1 addition (Fig. 1D). Concurrently, PAI-1 mRNA abundance increased within 2 h after initiation of TGF-beta 1 treatment and remained elevated at 24 h (Fig. 1D).

TGF-beta 1-induced TIMAP repression requires protein synthesis and histone deacetylase. In the presence of the RNA synthesis inhibitor actinomycin D, TIMAP mRNA abundance declined (Fig. 2A). When cells were treated concurrently with TGF-beta 1 and actinomycin D, the rate of decline of TIMAP mRNA abundance was similar to that observed with actinomycin D alone and also with TGF-beta 1 alone (Fig. 2A). These data suggest that the decline in TIMAP mRNA in response to TGF-beta 1 is not due to enhanced degradation.


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Fig. 2.   TGF-beta 1 decrease in steady-state TIMAP mRNA levels is not due to altered mRNA stability and depends on new protein synthesis and histone deacetylase. A: TGF-beta 1 regulates TIMAP mRNA by reducing transcription. TIMAP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA abundance was quantified densitometrically as a function of time, and density was expressed as TIMAP-to-GAPDH ratio. In untreated (control) cells, TIMAP mRNA abundance rose slightly over the 4 h. By contrast, in cells treated with actinomycin D, TIMAP mRNA was unstable and declined significantly. Rate of decline of TIMAP mRNA in the presence of TGF-beta 1 with or without actinomycin D was very similar to that observed with actinomycin D alone. B: cycloheximide (CHX) abolishes TGF-beta 1-induced decrease in TIMAP steady-state mRNA levels. Total RNA was collected from GEN cells treated with TGF-beta 1 for 6 h with or without CHX and analyzed by Northern blotting with probes for TIMAP, PAI-1, and rpL19. In the presence of CHX, the effect of TGF-beta 1 on TIMAP mRNA abundance is blocked, whereas stimulation of PAI-1 expression by TGF-beta 1 was still observed. C: histone deacetylase inhibition abolishes TGF-beta 1-mediated decrease in TIMAP steady-state mRNA levels. Total RNA was collected from GEN cells stimulated with or without TGF-beta 1 in the presence or absence of trichostatin A (TSA). No reduction in TIMAP mRNA level was observed in the presence of TSA. TSA did not alter TGF-beta 1-stimulated rise in PAI-1 mRNA.

To determine whether the effect of TGF-beta 1 on TIMAP requires new protein synthesis, GEN cells were treated with TGF-beta 1 for 6 h in the presence or absence of CHX, and total RNA was collected and analyzed by Northern blotting. CHX blocked TGF-beta 1-mediated suppression of TIMAP mRNA but did not inhibit TGF-beta 1 induction of PAI-1 transcription (Fig. 2B). The transcriptional repressors SnoN (34), Ski (41), and TGIF (40) interact with R-Smad-Co-Smad complex and recruit histone deacetylase (HDAC) into a multimeric repressor complex. We therefore examined whether TIMAP downregulation by TGF-beta 1 involves HDAC. GEN cells were treated with TSA, a potent and specific inhibitor of HDAC (42), for 30 min before addition of TGF-beta 1 to the culture. TSA alone had no effect on TIMAP mRNA levels. However, in the presence of TSA, TGF-beta 1 no longer repressed TIMAP expression (Fig. 2C), suggesting that an unidentified transcriptional repressor requiring HDAC is involved in TGF-beta 1 downregulation of TIMAP. In contrast to the findings for TIMAP, PAI-1 expression was induced to a similar extent by TGF-beta 1 in the absence and presence of TSA without superinduction (Fig. 2C).

TIMAP full-length cDNA and genomic structure. The full-length bTIMAP cDNA sequence is 6,299 bp long and encodes an ORF of 568 amino acids. The putative translation initiation codon was assigned on the basis that 1) there are no upstream ATGs, 2) a stop codon is present 11 codons upstream from the initiation codon, and 3) a Kozak consensus sequence (GCCatgG) (13-15) was found surrounding the initiation codon.

The full-length hTIMAP cDNA was obtained by first establishing that KIAA0823 (35) (accession no. AB020630, GenBank), originally isolated from human brain, represents a partial human cDNA homologous to the bTIMAP cDNA. The full-length cDNA of hTIMAP is 6,113 bp long and encodes a 567-aa polypeptide. The amino acid sequence of hTIMAP is 97% identical to that of bTIMAP. The predicted TIMAP protein contains strong 5' nuclear localization consensus sequences, 5 ankyrin repeats within its NH2-terminal half, a protein phosphatase-1 (PP1) binding domain just upstream of the first ankyrin domain, and a CAAX box (CRIS) at the COOH terminus (Fig. 3B).


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Fig. 3.   TIMAP is a member of a new family of CAAX box proteins. A: exon-intron organization of the TIMAP gene. B: human TIMAP (top) and mouse MYPT3 (bottom) amino acid sequences. Boxes enclose conserved regions; dashes represent gaps introduced to show alignment. a, Bipartite nuclear localization signal in TIMAP; b, protein phosphatase-1 (PP1) binding domain; c-g, ankyrin domains; h, prenylation site. *, Locations of potential tyrosine phosphorylation sites in TIMAP sequence. C: evolutionary distances for MYPT and PPP1R16B families of proteins. GenBank protein accession numbers are shown in parentheses. Hs, Homo sapiens, Bt, Bos taurus, Mm, Mus musculus; Ce, Caenorhabditits elegans; Dm, Drosophila melanogaster.

TIMAP belongs to a new gene family (KIAA0823) of which MYPT3 (32) (accession nos. XM035347 and AY010723, GenBank) is another well-characterized member. The amino acid identity between the predicted MYPT3 and TIMAP proteins is 44.7%. The Homo sapiens hypothetical protein MGC14333 (accession no. BC007854, GenBank) is a very close homolog or, potentially, a splicing variant of MYPT3. Similarities and conserved domains between TIMAP and MYPT3 are shown in Fig. 3B. Other members of the family include the Caenorhabditits elegans and Drosophila melanogaster KIAA0823 homologs (accession nos. Z75544 and AE003519, respectively, GenBank). All KIAA0823 family members contain a COOH-terminal prenylation motif, conserved ankyrin domains, and a conserved PP1 binding site. MYPT3 was previously noted to share domain homology for ankyrin and PP1 binding domains with the MYPT1 and MYPT2 proteins; however, MYPT1 and MYPT2 do not contain a prenylation motif. Evolutionary distances for the KIAA0823 and MYPT families (Fig. 3C) were established using the split decomposition approach (3, 9). The gene name proposed by others to the Human Genome Organization is PPP1R16B [PP1, regulatory (inhibitor) subunit 16B] on the basis of domain homology with MYPT3. Hence, the family is referred to by that name in Fig. 3C.

The genomic structure of the hTIMAP gene was deduced from three overlapping human genomic sequences reported in GenBank that match hTIMAP cDNA (accession nos. AL031657, AL121889, and AL023803, GenBank). The hTIMAP gene is located on chromosome 20q11.22-12, and it spans >87 kbp. hTIMAP mRNA is encoded by 10 exons, and perfect intron/exon splice motifs were identified at the intron/exon boundaries (Fig. 3A).

TIMAP mRNA expression. All cultured endothelial cells (bovine GEN and BAE cells and human aorta, umbilical vein, and microvascular endothelial cells) examined so far express TIMAP mRNA. TIMAP transcript was also observed in several hematopoietic cell lines (KG-1a, Molt4, Dami, and TF1; Fig. 4A). However, TIMAP was not detected in HeLa cells, WM793 melanoma cells, and HCT116 colorectal cells (Fig. 4A). In HEK-293 cells, a human embryonic kidney cell line, two transcripts larger than the TIMAP mRNA observed in other cells hybridized with the TIMAP cDNA probe. Whether these mRNAs are derived from the TIMAP gene, with alternate transcription start sites, or represent homologous mRNA not derived from the TIMAP gene remains to be determined.


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Fig. 4.   Steady-state levels of TIMAP mRNA in selected human cell lines and rat organs. A 271-bp human TIMAP (hTIMAP) cDNA fragment derived from the open reading frame was used as the probe for both blots. A: 6.2-kb TIMAP mRNA was observed in all human hematopoietic cell lines examined, not in epithelial cells (see text for details). In HEK-293 cells, two mRNAs differing in size from the TIMAP mRNA were observed. Whether these represent mRNA derived from the TIMAP gene containing unidentified exons or homologous mRNA derived from a different gene remains to be determined. B: TIMAP mRNA abundance in rat organs. TIMAP mRNA was observed in all tissues derived from the central nervous system, as well as lung, spleen, kidney, testis, and adrenal, with low or absent signal in eye, heart, liver, and skeletal muscle.

Using a fragment of the hTIMAP cDNA as a probe, we analyzed RNA from various rat organs. TIMAP mRNA of ~6.2 kb was expressed in all tissues from the central nervous system (CNS). The TIMAP transcript was also highly expressed in lung, spleen, kidney, and testis (Fig. 4B), but its expression was low or undetectable in heart, liver, and smooth muscle. Transcripts of the size observed in the HEK-293 cells were not observed in tissues in vivo.

SAGE libraries afford an added level of evaluating gene expression in cells and tissues (16, 38) (www.ncbi.nlm.nih.gov/SAGE). The SAGE tag for hTIMAP is TCCCTGGAGT. In 95 public SAGE tag libraries, the TIMAP tag was found in 6 libraries: 4 prepared from bulk tissue mRNA (normal thalamus, normal prostate, medulloblastoma, and astrocytoma tumors) and 2 prepared from cultured human microvascular endothelial cells. The TIMAP SAGE tag was not found in 47 SAGE tag libraries prepared from cultured human nonendothelial cell lines. In a SAGE tag library of normal endothelial cells isolated freshly from colon, the TIMAP tag was also expressed, while it was absent from endothelial cells isolated from colonic carcinoma (33). Overall, in all endothelial cell SAGE libraries available to us, the TIMAP tag frequency was 25.9/106 tags (7 TIMAP tags, 4 libraries, 270,059 tags). The frequency for the same tag was 0.8/106 cells in all nonendothelial cell libraries (5 TIMAP tags, 93 libraries, 3,778,264 tags; P < 0.001 by chi 2 analysis). These data are consistent with much higher levels of TIMAP expression in endothelial cells than in other cell types.

In vivo, TIMAP is expressed predominantly in endothelial cells. Two distinct polyclonal anti-TIMAP peptide antibodies resulted in essentially identical staining patterns in vivo. In newborn kidney, the TIMAP antibodies localized predominantly to the vasculature, with strong staining of endothelial cells in arteries and arterioles (Fig. 5, A and B). Dual-immunofluorescence studies demonstrated that staining was excluded from alpha -smooth muscle actin-positive cells (Fig. 5, C and D). Specific staining of the renal vasculature without staining of renal parenchymal epithelial cells is shown in Fig. 5D. In adult kidney and heart, TIMAP antibodies similarly localized to blood vessels (not shown).


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Fig. 5.   TIMAP protein is expressed in vascular endothelial cells. Kidneys from 5-day-old rats were stained with polyclonal rabbit anti-TIMAP peptide antibodies. A and B: immunolocalization of anti-TIMAP antibodies was restricted to the vasculature, including glomeruli. In large vessels (A), TIMAP antibodies localize to the endothelium. C and D: rabbit polyclonal anti-TIMAP (red) and mouse monoclonal alpha -smooth muscle (green) do not colocalize in large arteries (C) or in renal arterioles (D), and TIMAP antibodies label the inner lumen of the vessels consistent with endothelial expression. Arrows (B and D) indicate glomerular labeling. E: single-label immunofluorescence labeling of neonatal kidney with TIMAP antibody shows predominantly vascular staining and absence of label from the parenchyma.

TIMAP is a novel CAAX box protein localized at the plasma membrane. TIMAP contains a CAAX box at the COOH terminus and also contains another cysteine within 12 amino acids of the CAAX box. This suggests that TIMAP is a novel CAAX box protein subject to prenylation and plasma membrane localization. Two mammalian expression vectors were prepared: one encoding a green fluorescent protein (GFP)-TIMAP fusion protein and the second a GFP-TIMAP(C564S) fusion protein in which the cysteine in the CAAX box was replaced by a serine by site-directed mutagenesis. This mutation has previously been shown to abolish membrane association of CAAX box proteins (8, 12, 25). The GFP-TIMAP fusion proteins were then expressed transiently in GEN, COS-7, and MDCK cells. Data for MDCK and GEN cells are shown in Fig. 5. Identical results were obtained from experiments with COS-7 cells (not shown). At 24 h after transfection, control GFP not fused to TIMAP was diffusely expressed in all cell types, tending to localize most strongly to the nucleus (Fig. 6, A and D). The GFP-TIMAP fusion protein was expressed at the cell membrane and in the perinuclear region (Fig. 6, B and E). By contrast, the GFP-TIMAP(C564S) fusion protein did not localize to the plasma membrane (Fig. 6, C and F). These results suggest that, similar to other CAAX box proteins, TIMAP localizes to the cell membrane and that membrane localization requires posttranslational prenylation at the COOH terminus. In MDCK (Fig. 6F) and COS-7 (not shown) cells, TIMAP(C564S) was also found in the nucleus, consistent with the NH2-terminal nuclear localization signal.


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Fig. 6.   Membrane localization of TIMAP requires the CAAX motif. GEN (a-c) and Madin-Darby canine kidney (MDCK, d-f) cells were transiently transfected with expression plasmids for enhanced green fluorescent protein (EGFP, a and d), EGFP-TIMAP fusion protein (b and e), or EGFP-TIMAP(C564S) fusion protein (c and f). Solid arrows, membrane localization of EGFP-TIMAP fusion protein in both cell types. EGFP-TIMAP(C564S) mutant fusion protein did not localize to plasma membrane but is seen in large cytoplasmic inclusions in both cell types. In MDCK cells, punctate accumulation of EGFP-TIMAP(C564S) fusion protein was also found in nucleus (open arrow).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study describes the initial characterization of a novel gene and its protein product TIMAP, also referred to as PPP1R16B. The TIMAP gene, located on human chromosome 20q11.22-12, contains 10 exons and spans >87 kb. Its mRNA is highly expressed in cultured endothelial and hematopoietic cells and in the CNS, adrenal, lung, and spleen in vivo. The TIMAP protein is highly expressed in vascular endothelial cells in vivo. Steady-state TIMAP mRNA levels in endothelial cells decrease significantly in response to TGF-beta 1, an effect that appears to be due to a protein synthesis- and HDAC-dependent reduction in transcription. The predicted TIMAP protein contains strong NH2-terminal nuclear localization signals, a PP1-interacting domain, five ankyrin repeats in its NH2-terminal half, and a COOH-terminal CAAX box motif. When expressed transiently in endothelial and epithelial cells, the wild-type TIMAP protein localizes to the plasma membrane, and a point mutation in the CAAX box, predicted to eliminate COOH-terminal prenylation, abolishes membrane localization. Hence, TIMAP is a novel, TGF-beta 1-regulated CAAX box protein highly expressed in endothelial and hematopoietic cells in vitro and in vascular endothelium and the CNS in vivo.

The TIMAP cDNA sequence was found by RDA screening of glomerular capillary endothelial cells for unknown transcripts subject to regulation by TGF-beta 1. The RDA product TIMAP was further characterized, because a previously identified homolog, the 5,597-bp partial human cDNA KIAA0823 (35) (accession no. AB020630, GenBank) predicted a COOH-terminal CAAX box, suggesting that TIMAP could represent a novel signaling molecule. The predicted ORFs for hTIMAP and bTIMAP are 1,701 and 1,704 bp long, respectively, and both cDNAs contain long (4,411 and 4,407 bp, respectively) 3'-untranslated regions. The predicted proteins are 568 (bovine) and 567 (human) amino acids long, and the amino acid sequences are highly conserved (97% homologous) between the two species. Three overlapping human genomic clones that map to chromosome 20q11.22-12 were found which contain the entire TIMAP cDNA as 10 exons. The total genomic span of the TIMAP sequence is just over 87 kb. Whether alternate upstream exons could contribute to larger transcripts, such as those observed in HEK-293 cells (Fig. 4A), is unknown.

One conserved motif found in the predicted TIMAP protein sequence is a CAAX box (CRIS) at its COOH terminus. The CAAX motif, always located at the COOH terminus, predicts posttranslational modification through prenylation, which then tends to target the protein to the inner leaflet of the plasma membrane (8). The prenylation of CAAX box proteins renders their COOH terminus highly hydrophobic and, thus, capable of interacting with the membrane phospholipid bilayers. The finding that the terminal amino acid of the putative TIMAP protein is serine would predict that it is a substrate for a farnesyltransferase (8).

The CAAX motif is found in a restricted set of protein families, among them Ras family members, nuclear lamins, and the gamma -subunit of trimeric G proteins (8). Mutations of the cysteine residue, to prevent prenylation, interfere with membrane localization of CAAX box proteins and, in some cases, also with other protein-protein interactions (12). In the case of TIMAP, the wild-type protein localized to the plasma membrane of endothelial and epithelial cells when transiently expressed (Fig. 6). The C564S point mutation in which cysteine is replaced with serine in the CAAX box of TIMAP resulted in loss of this membrane localization and retention of the protein in large cytoplasmic inclusions (Fig. 6, c and f). Hence, the functional significance of the CAAX box, at least for membrane localization of TIMAP, has been established. In COS-7 and MDCK cells, but not in endothelial cells, transiently expressed GFP-TIMAP(C564S) fusion protein was found in the nucleus. It is likely that nuclear localization is mediated by the nuclear localization sequences found in the TIMAP NH2 terminus (Fig. 3B). The difference in the ability of TIMAP(C564S) to localize to the nucleus in epithelial vs. endothelial cells is not understood.

The ORF of the TIMAP sequence also predicts five ankyrin repeats in its NH2-terminal half. Ankyrin repeats generally mediate protein-protein interactions. There is a large diversity of ankyrin repeat-containing proteins with many different intracellular locations and functions (31). To our knowledge, no other family of proteins containing ankyrin repeats and CAAX box has been described.

The TIMAP protein shares significant domain homology with a recently cloned protein named MYPT3. At the amino acid level, there is 44.7% homology between TIMAP and MYPT3. The MYPT3 protein was discovered by the yeast two-hybrid approach using PP1 as the bait (32). MYPT3 was shown to bind PP1 and to have inhibitory activity toward this phosphatase. MYPT3 functions are similar to those of two other proteins, MYPT1 and MYPT2, which share the ankyrin and PP1 binding domains with MYPT3 and TIMAP (Fig. 3B). However, because the MYPT1 and MYPT2 proteins do not share the CAAX box found in the PPP1R16B family of proteins, they are more distantly related to TIMAP than is MYPT3 (Fig. 3C).

The TIMAP transcript abundance in endothelial cells declines within 2-4 h in after addition of TGF-beta 1 to the cells (Figs. 1D and 2A). When new RNA synthesis was blocked with actinomycin D, the TIMAP mRNA level declined at a rate very similar to that observed in cells treated with TGF-beta 1 alone or with a combination of TGF-beta 1 and actinomycin D (Fig. 2A). These findings suggest that the effect of TGF-beta 1 on TIMAP mRNA abundance does not reflect a change in TIMAP mRNA stability. Inasmuch as TGF-beta 1 did not alter TIMAP transcript levels in the presence of CHX, it is evident that TGF-beta 1-mediated regulation of TIMAP mRNA levels is indirect and requires new protein synthesis. TGF-beta 1-dependent transcriptional repression involving proteins that assemble in multimeric complexes with HDAC is now well described. The proteins Ski, SnoN, and TGIF can associate with activated Smad complexes, resulting in recruitment of corepressors and HDAC to Smad-responsive promoters (34, 40, 41). In this study, TGF-beta 1-mediated reduction in TIMAP transcript abundance was sensitive to TSA, a potent inhibitor of HDAC. The dependence of TIMAP mRNA downregulation on HDAC could reflect transcriptional repression of TIMAP.

We observed abundant expression of TIMAP in cultured bovine and human endothelial cells derived from large and small blood vessels. The TIMAP SAGE tag was found in the two public human endothelial cell SAGE libraries and in a normal colon endothelial cell library (33) at a frequency >30-fold higher than its expression frequency in all public nonendothelial libraries (~4 × 106 tags) combined. These findings suggested that TIMAP is highly expressed in endothelial cells. Two distinct polyclonal peptide antibodies representing TIMAP epitopes not found in MYPT3 or other known or predicted protein sequences localized predominantly to the vasculature. In blood vessels examined by dual-label immunofluorescence with TIMAP and alpha -smooth muscle actin antibodies, TIMAP was excluded from the smooth muscle cell layer, confirming the endothelial cell location. Hence, SAGE expression surveys, Northern blot analysis of cultured cells, and histology confirm that TIMAP is expressed in endothelial cells.

We also observed TIMAP mRNA expression in a number of transformed hematopoietic cell lines. There are no reported SAGE tag libraries for hematopoietic cell lines; hence, no further information on the level of TIMAP expression in cells of that lineage could be obtained from that source. Because endothelial and hematopoietic cells are derived from common precursors, it is tempting to speculate that TIMAP may be functionally important in cells derived from the hemangioblast lineage.

We did observe expression of TIMAP mRNA in several rat tissues, including all portions of the CNS examined. In this regard, it is also of note that the KIAA0823 cDNA was initially cloned from a human brain cDNA library (35). Furthermore, bulk medulloblastoma and astrocytoma and bulk thalamus SAGE libraries contained the TIMAP tag. It therefore seems likely that TIMAP is also expressed in the CNS. Clearly, more work will need to be done to further define the localization of TIMAP expression in vivo.

In conclusion, we describe the cDNA and putative amino acid sequence of a novel CAAX box protein TIMAP, the CAAX box mediating localization of transiently expressed protein, to the plasma membrane. The TIMAP protein is predicted to contain NH2-terminal nuclear localization signals, multiple ankyrin repeats, and a PP1 binding domain. To our knowledge, the PPP1R16B family of proteins, of which TIMAP and MYPT3 are members, is the first to contain the combination of multiple ankyrin repeats and CAAX motif. The TIMAP mRNA is highly expressed in endothelial and hematopoietic cells in culture and in vascular endothelium in vivo. We postulate that transcriptional repression of TIMAP may be an important component of the apoptotic and/or capillary morphogenesis response of endothelial cells to TGF-beta 1.


    ACKNOWLEDGEMENTS

The electronic SAGE libraries for normal and tumor endothelium from colon were kindly provided by B. Vogelstein. V. Senchak provided expert cell culture assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-50764 (B. J. Ballermann) and HL-56091 (B. J. Ballermann and C. J. Lowenstein). W. Cao was supported by the Maryland Chapter of the National Kidney Foundation.

Address for reprint requests and other correspondence: B. J. Ballermann, Albert Einstein College of Medicine, Ullmann Bldg. 619, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: bjballer{at}aecom.yu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 27, 2002;10.1152/ajpcell.00442.2001

Received 14 September 2001; accepted in final form 18 February 2002.


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