Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M210376200 on October 18, 2002

J. Biol. Chem., Vol. 277, Issue 52, 50788-50794, December 27, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/52/50788    most recent
M210376200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mo, J.
Right arrow Articles by Blobe, G. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mo, J.
Right arrow Articles by Blobe, G. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Regulation of ALK-1 Signaling by the Nuclear Receptor LXRbeta *

Jinyao MoDagger , Shijing J. FangDagger , Wei Chen§, and Gerard C. BlobeDagger

From the Dagger  Departments of Medicine and Pharmacology and Cancer Biology and § Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, October 10, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The transforming growth factor beta  (TGF-beta ) receptor, ALK-1, is expressed specifically on endothelial cells and is essential for angiogenesis, as demonstrated by its targeted deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia. Although ALK-1 and another endothelial-specific TGF-beta receptor, endoglin, both bind TGF-beta with identical isoform specificity and form a complex together, neither has been shown to signal in response to TGF-beta , and the mechanism by which these receptors signal in endothelial cells remains unknown. Here we report the identification of the nuclear receptor liver X receptor beta  (LXRbeta ) as a modulator/mediator of ALK-1 signaling. The cytoplasmic domain of ALK-1 specifically binds to LXRbeta in vitro and in vivo. Expression of activated ALK-1 results in translocation of LXRbeta from the nuclear compartment to the cytoplasmic compartment. The interaction of activated ALK-1 with LXRbeta in the cytoplasmic compartment results in the specific phosphorylation of LXRbeta by ALK-1, primarily on serine residues. LXRbeta subsequently modulates signaling by ALK-1 and the closely related TGF-beta receptor, ALK-2, as demonstrated by specific and potent inhibition of ALK-1- and ALK-2-mediated transcriptional responses, establishing LXRbeta as a potential modulator/mediator of ALK-1/ALK-2 signaling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor beta  (TGF-beta )1 is a member of a family of dimeric polypeptide growth factors that regulate cellular proliferation and differentiation as well as the processes of embryonic development, wound healing, and angiogenesis in a cell- and context-specific manner (1). Mutations in TGF-beta receptors or their intracellular signaling molecules have been described in association with tumorigenesis and the human disease hereditary hemorrhagic telangiectasia (HHT). HHT is an autosomal dominant disease in which the primary defect is vascular dysplasia resulting in telangiectasia and arteriovenous malformations. Clinically this disorder results in telangiectases of the skin, recurrent epistaxis, and gastrointestinal bleeding as well as shunting phenomena and neurological sequela because of arteriovenous malformations in the pulmonary and central nervous system (2). The pathological lesions of HHT consist of dilated vessels, lined by a single layer of endothelium attached to a continuous basement membrane, that are thought to form from dilation of postcapillary venules. Genetic linkage studies of families with HHT identified the genes for two receptors in the TGF-beta family, endoglin and ALK-1 (a type I receptor in the TGF-beta family) as the genes responsible for hereditary hemorrhagic telangiectasia-1 and -2, respectively (3, 4). Endoglin and ALK-1 are expressed specifically on endothelial cells, and endoglin production is increased in proliferating endothelium (5). Mice lacking endoglin or ALK-1 died at embryonic day 10.5-11.5 because of defects in angiogenesis (6-10). Histochemical studies of the embryos revealed primary defects in the endothelial and smooth muscle cells of the developing blood vessels, confirming an essential role for these TGF-beta receptors in angiogenesis.

ALK-1 and endoglin mutations in HHT result in similar phenotypes: both have similar affinity and specificity for TGF-beta (with preference for the TGF-beta 1 and -3 isoforms) and have been shown to associate with one another, suggesting that both receptors transduce similar signals essential for endothelial cell function (11). However, the nature of the signaling pathway downstream from these receptors is unknown.

To establish the signaling pathway downstream of ALK-1 and endoglin, yeast two-hybrid screens utilizing the cytoplasmic domains of these receptors were performed. Here we report the identification of the nuclear receptor LXRbeta as a protein that specifically binds to the cytoplasmic domain of activated ALK-1. ALK-1 alters LXRbeta cellular localization and phosphorylates LXRbeta , primarily on serine residues, and LXRbeta is able to modulate ALK-1 signaling, establishing LXRbeta as a potential member of the ALK-1 signaling pathway.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screens-- The mating system of James, Halladay, and Craig was utilized (12). Briefly, a human lung library (Clontech) was screened with baits composed of the cytoplasmic domain of ALK-1 and mutants of the cytoplasmic domain of ALK-1 (ALK-1-Q200D and ALK-K229R) cloned into pGBD, in-frame with the Gal4 DNA binding domain. Yeast containing bait and the prey library cloned in pGAD, in-frame with the Gal4 activation domain, were mated overnight in YPAD (yeast extract, peptone, adenine, and dextrose) media at 30 °C, plated on Trp-, Leu-plates and incubated at 30 °C for 3-5 days to select for yeast containing both bait and prey vectors. Colonies were replica-plated on His- or His-, Ade- plates to assay for interaction as assessed by growth under these conditions. Interacting clones were tested for ability to repeat, bait, and prey dependence and specificity with other baits (lamin, myc, endoglin, ALK-2) to screen out false positives. Clones that passed these screens were sequenced and further analyzed.

Yeast Two-hybrid Interaction Assay-- Appropriate strains of yeast (a strain for bait, alpha  strain for library) were transformed with pGBD control vector or pGBD-ALK-1, -ALK-1Q-D, -ALK-1K-R, -ALK-2, or -END (containing the cytoplasmic domain of these respective proteins) and pGAD-LXRbeta (encoding LXRbeta lacking the first 14 amino acids). These yeast were mated overnight in YPAD media at 30 °C, plated on Trp-, Leu- plates, and incubated at 30 °C for 3-5 days to allow diploid cells to form visible colonies. Colonies were replica-plated on His-, Ade- plates to assay for interaction as indicated by colony growth.

GST Affinity Binding Assay-- COS-7 or 293T cells expressing HA- or FLAG-tagged LXRbeta or HA-tagged LXRalpha were lysed with 1% Triton X-100 lysis buffer and precleared with glutathione-agarose beads. Equal amounts of cell lysate were incubated with GST fusion proteins of the cytoplasmic domain of ALK-1 (GST-ALK-1), activated ALK-1 (GST-ALK-1Q-D), or GST alone complexed with glutathione-agarose beads. The beads were harvested by centrifugation and washed three times with lysis buffer. Binding proteins were analyzed by SDS-PAGE and Western blot analysis with alpha HA or alpha FLAG antibody as appropriate.

Co-immunoprecipitation Assay-- THP-1 macrophage cells were lysed in RIPA lysis buffer with 1 mM phenylmethylsulfonyl fluoride and 0.02% leupeptin. The lysates was precleared by centrifugation, and 500-µl aliquots were incubated with polyclonal preimmune serum, polyclonal alpha ALK-1 antibody (made to the cytoplasmic domain of ALK-1, peptide KREPVVAVAAPASSESSST), or alpha LXR antibody (Affinity BioReagents) for 2 h at 4 °C. The immune complexes were harvested by addition of Protein A beads. The pellets were washed three times with ice-cold lysis buffer. 50 µl of 2× sample buffer was added, and samples were boiled and loaded on a 12.5% SDS-PAGE, transferred to nitrocellulose, and visualized by Western blot analysis with alpha ALK-1 antibody.

Confocal Microscopy-- Transfected HEK-293 cells were split onto collagen-coated 35-mm plastic dishes with glass bottoms (MatTek, Ashland, MA) and cultured overnight. GFP images were generated and collected using single line excitation (488 nm) and a 505-nm emission filter with a Zeiss laser scanning confocal microscope (LSM-510).

Phosphorylation Assays-- COS-7 cells were transfected with various TGF-beta receptors and HA-tagged LXRbeta or -alpha . After 48 h, the cells were washed in phosphate-free medium and labeled with [32P]orthophosphate (1.0 mCi/ml) for 4 h. Cells were washed with phosphate-buffered saline, lysed with RIPA lysis buffer, immunoprecipitated with alpha HA antibody and Protein G-Sepharose, and analyzed on 10% SDS-PAGE gels; phosphorylation was detected by phosphoimager analysis of the dried gels.

Phosphoamino Acid Analysis-- In vivo 32P-labeled LXRbeta was immunoprecipitated with alpha HA antibody, resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was washed, and the 32P-labeled LXRbeta was detected by autoradiography and excised from the membrane. The protein was hydrolyzed to completion by placing in 6 M HCl at 110 °C for 1 h, dried down, resuspended, and spotted on a TLC plate along with unlabeled phosphoamino acid standards. The TLC was wet with 66% (pH 1.9, electrophoresis buffer (0.58 M formic acid, 1.36 M acetic acid)): 34% (pH 3.5, electrophoresis buffer (0.5% (v/v) pyridine, 0.87 M acetic acid, 0.5 mM EDTA)) and electrophoresed for 1 h at 1.5 kV. The plate was dried at 50 °C, sprayed with 0.25% ninhydrin in acetone, reheated to visualize the standards, and then analyzed by phosphoimager analysis.

Phosphopeptide Mapping-- In vivo 32P-labeled LXRbeta was resolved by SDS-PAGE, detected by autoradiography, and excised from the dried gel. The gel was rehydrated in 50 mM ammonium bicarbonate, pH 7.3, and the proteins were serially extracted by addition of 1% 2-mercaptoethanol and 0.1% SDS until over 60% of labeled protein was extracted by Cerenkov counting. 20 µg of Rnase (carrier protein) was added; the mixture was trichloroacetic acid-precipitated, oxidized in cold performic acid, dried, and digested overnight in 50 mM ammonium bicarbonate, pH 8.0. with 10 µg of trypsin. The digested peptides were lyophilized, resuspended in pH 1.9 electrophoresis buffer: pH 3.5 electrophoresis buffer (2:1), and spotted on a TLC plate. The TLC was electrophoresed in the first dimension for 20 min at 1.0 kV, then air dried, rotated 90° counterclockwise, and separated in the second dimension by chromatography in standard chromatography buffer until the buffer was 2 cm from the top of plate. The plate was dried at 65 °C and analyzed by phosphoimager analysis.

Luciferase Reporter Gene Assays-- P19 or L6 cells were plated onto 24-well plates at 20,000 cells/ml 1 day prior to transfection. The cells were transiently transfected using FuGene 6 reagent (Roche) with 300 ng/well of total DNA (100 ng of XVent-Lux or pE2.1-Lux, 100 ng of LXRbeta or -alpha expression vectors, and 100 ng of ALK-1 Q-D or -2 Q-D expression vectors). The following morning the cells were washed twice in phosphate-buffered saline and were immediately incubated in alpha -MEM (minimum essential medium) with 0.2% fetal bovine serum for 18-24 h at 37 °C. Afterward, cells were lysed in 100 µl of passive lysis buffer (Promega) and 25 µl used for luciferase assays (Promega). Data are from experiments done in triplicate and are presented as the mean -fold stimulations (± S.E.) of the luciferase induced by ALK-1 Q-D or -2 Q-D relative to samples transfected with control vector.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interaction of LXRbeta with ALK-1 in Vitro and in Vivo-- To elucidate potential downstream signaling molecules for ALK-1, the cytoplasmic domain of ALK-1 was utilized as a bait to screen for interacting proteins with the yeast two-hybrid system of James, Halladay, and Craig (12). Initial screens of a murine embryonic library (day 10.5) and a human lung library with the entire cytoplasmic domain of ALK-1 did not yield any specific clones, whereas screens with the entire cytoplasmic domain of ALK-2 done in parallel revealed numerous clones of FKBP12 as has been previously reported (13) (data not shown). Because the cytoplasmic domain of ALK-1 contains a serine/threonine protein kinase and constitutively active ALK-1 has been reported to activate BMP-responsive promoters (14, 15) and phosphorylate Smad1 and Smad5 (14, 15), baits of the cytoplasmic domain of constitutively active ALK-1 (ALK-1 Q200D, mutating glutamine 200 to aspartic acid in the GS domain) and kinase-dead ALK-1 (ALK-1 K229R, mutating lysine 229 to arginine in the ATP binding site) were utilized to rescreen the human lung library. The ALK-1 K229R bait again did not yield any specific clones; however, the screen with the ALK-1 Q200D bait yielded 17 specific clones. Three of these isolates encoded two separate clones, both encompassing nearly the entire open reading frame of the nuclear receptor LXRbeta (Fig. 1A). LXRbeta has been previously identified as an orphan nuclear receptor that forms obligate heterodimers with the retinoid X receptor to bind and regulate promoters with a unique hormone response element (LXRE) (16-20). LXRbeta contains an amino-terminal PEST domain followed by a DNA binding domain and then a ligand binding domain containing an activation function (AF-2) (Fig. 1A). More recently the oxysterols 24(S), 25-epoxycholesterol and 24(S) hydroxycholesterol, have been identified as potential endogenous ligands for LXRbeta and the closely related nuclear receptor LXRalpha (21, 22). In addition, LXRalpha has been established as a sensor of dietary cholesterol, consistent with its restricted expression in tissues rich in lipid metabolism (23). In contrast, the role of LXRbeta , which is ubiquitously expressed, remains to be established. Significantly, unlike LXRalpha knockout mice, LXRbeta knockout mice lack a metabolic/lipid metabolism phenotype (24). Association of ALK-1 Q200D with LXRbeta was specific because LXRbeta was not isolated in screens with ALK-1, -1 K229R, or -2 (data not shown). In addition, the cytoplasmic domain of ALK-1 Q200D could interact with LXRbeta in the yeast two-hybrid interaction assay, whereas the cytoplasmic domains of ALK-1, -1 K229R, -2, or endoglin could not (Fig. 1B).


View larger version (49K):
[in this window]
[in a new window]
  		Fig. 1.   ALK-1 specifically interacts with LXRbeta in vitro and in vivo. A, the structure of full-length LXRbeta , including the DNA binding domain (DNA BD) and the ligand binding domain (ligand BD) containing an activation factor 2 (AF-2) domain is depicted (top), with the regions encoded by two distinct yeast two-hybrid cDNA clones isolated as binding partners for ALK-1 indicated by bold lines along the initial amino acid (aa) of the clone (below). B, yeast expressing Gal4 DNA binding domain fusion proteins of the cytoplasmic domains of ALK-1, ALK-1 Q-D, ALK-1 K-R, ALK-2, and endoglin (END), or Gal4 DNA binding domain alone (GBD) (baits) were mated with yeast expressing the Gal4 activation domain fused to LXRbeta (prey) and plated on Trp-, Leu- plates to select for yeast containing both bait and prey and then on His-, Ade- plates and assessed for growth, indicative of an interaction between bait and prey proteins in the yeast two-hybrid interaction assay. C, GST alone or GST fusion proteins of the cytoplasmic domain of ALK-1 or ALK-1 Q-D were incubated with equal amounts of lysate from COS-7 cells expressing HA- or FLAG-tagged LXRbeta (lanes 5-8) or HA-tagged LXRalpha (lanes 1-4) as indicated. GST or GST fusion proteins were pulled down by glutathione beads and total lysate (lanes 1 and 5) or proteins pulled down with GST (lanes 2 and 6), or the GST fusion proteins (lanes 3, 4, 7, 8) were analyzed on SDS-PAGE followed by immunoblot analysis with alpha HA or alpha FLAG antibody as indicated. Coomassie staining of the GST and GST fusion proteins utilized in the pull-down assays is shown in the bottom panel. D, THP-1 cells were lysed in RIPA lysis buffer, immunoprecipitated by preimmune serum, alpha ALK-1, or alpha LXR antibodies as indicated, and the immunoprecipitates analyzed on SDS-PAGE followed by immunoblot analysis with alpha ALK-1 antibody. Data shown are representative of three independent experiments.

To confirm the interaction of ALK-1 with LXRbeta , GST fusion proteins of the cytoplasmic domain of ALK-1 and -1 Q200D and an HA-tagged LXRbeta lacking the first 14 amino acids of the coding sequence (corresponding to the longer yeast two-hybrid clone isolated, Fig. 1A) were made. HA-tagged LXRbeta was efficiently expressed in COS-7 cells and was specifically pulled down by GST-ALK-1 Q200D as well as by GST-ALK-1 (Fig. 1C, top panel, lanes 7 and 8) but not by GST alone (Fig. 1C, lane 6). The interaction between ALK-1 and LXRbeta in the GST affinity binding assay was unexpected because this interaction could not be detected in the yeast two-hybrid system (Fig. 1B), suggesting that ALK-1 may be activated in mammalian cell extracts. To further investigate the interaction of ALK-1 and LXRbeta , a FLAG-tagged (amino-terminal) full-length LXRbeta was made and utilized in the GST affinity binding assay. FLAG-tagged LXRbeta was efficiently expressed in 293T cells and specifically pulled down by GST-ALK-1 Q200D (Fig. 1C, second panel, lane 8) but not by GST-ALK-1 or GST alone (Fig. 1C, lanes 6 and 7), suggesting that the first 14 amino acids of LXRbeta may regulate the interaction of ALK-1 and LXRbeta . Next, the interaction of ALK-1 with the closely related nuclear receptor, LXRalpha , was assessed. Neither GST-ALK-1 Q200D nor -ALK-1 was able to pull down LXRalpha (Fig. 1C, top panel, lanes 3 and 4). Taken together these results confirm a specific interaction between ALK-1 and LXRbeta in vitro.

To determine the regions important for the interaction between ALK-1 and LXRbeta , we examined the sequences of each protein for potential interaction motifs. This examination revealed an LXXLL motif in the cytoplasmic domain of ALK-1 (amino acids 174-178) known to mediate interaction of proteins with the AF-2 domain of nuclear receptors (25). To establish whether this LXXLL motif was responsible for mediating interaction of ALK-1 with the AF-2 of LXRbeta , the LXXLL motif was mutated in GST-ALK-1 Q200D and the mutant assessed for interaction with full-length LXRbeta . Mutation of LXXLL (LGDLL to LAAAA) of ALK-1 did not alter the ability of ALK-1 Q200D to bind LXRbeta (data not shown). In addition, mutation or deletion of the AF-2 of LXRbeta did not alter the ability of LXRbeta to bind ALK-1 Q200D (Fig. 1C, third panel, lanes 7 and 8, data not shown), confirming that these were not the regions responsible for interaction of ALK-1 and LXRbeta . We then analyzed the ability of specific regions of LXRbeta to bind to ALK-1 Q200D in vitro. These studies revealed that the ligand binding domain of LXRbeta (amino acids 286-461) was unable to bind ALK-1 Q200D in vitro (data not shown), suggesting that the binding motif resides in the first 286 amino acids of LXRbeta . Attempts to directly confirm binding of amino acids 1-286 of LXRbeta containing the DNA binding domain were unsuccessful because constructs of LXRbeta (amino acids 1-286) could not be stably expressed (data not shown).

Finally, to determine whether this interaction occurred in vivo and without overexpression of ALK-1 and LXRbeta (i.e. with physiological levels of expression of ALK-1 and LXRbeta ), we screened cells known to express LXRbeta for expression of ALK-1. This survey revealed that the THP-1 macrophage cell line known to express LXRbeta (26) also expressed ALK-1 (Fig. 1D, lane 2). Therefore, we used the THP-1 cell line to perform co-immunoprecipitation studies with antibodies to LXRbeta and ALK-1. An antibody to LXRbeta was able to co-immunoprecipitate ALK-1 in THP-1 cell lysates (Fig. 1D, lane 3), whereas preimmune serum was not (Fig. 1D, lane 1). Conversely, an antibody to ALK-1 co-immunoprecipitated LXRbeta in THP-1 cell lysates (data not shown). Taken together, these findings strongly suggest a physiological interaction between ALK-1 and LXRbeta .

Localization and Co-expression of LXRbeta and ALK-1-- Because the nuclear receptor LXRbeta is thought to reside primarily in the nucleus and ALK-1 is a membrane-bound receptor, we explored how these receptors might interact in vivo. To address this issue, we examined the localization of LXRbeta in vivo. We constructed and expressed a GFP fusion protein of LXRbeta in HEK-293 cells with or without ALK-1 Q-D and examined localization of GFP-LXRbeta in live cells by confocal microscopy. In the absence of ALK-1, GFP-LXRbeta was predominately, but not exclusively, expressed in the nucleus in most cells (Fig. 2, A and B), whereas GFP alone was uniformly distributed between the nucleus and cytoplasm as previously reported (27) (data not shown), and beta -arrestin 2-GFP was expressed exclusively in the cytoplasm (Fig. 2G). However, in some cells in the absence of ALK-1 (~5%), GFP-LXRbeta was more uniformly distributed between the nucleus and cytoplasm (Fig. 2C). In the presence of ALK-1 Q-D, localization was altered so that GFP-LXRbeta was predominately expressed in the nucleus in only a minority of cells (~20%), (Fig. 2D), whereas in the majority of cells, GFP-LXRbeta was uniformly distributed between the nucleus and cytoplasm (Fig. 2E). In addition, in some cells (~5%), a predominant membrane localization of GFP-LXRbeta was observed (Fig. 2F, arrow), suggesting that ALK-1 Q-D results in a nuclear to cytoplasmic translocation of LXRbeta and that ALK-1 has access to LXRbeta in the cytoplasm.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2.   ALK-1 stimulates translocation of LXRbeta from the nuclear to cytoplasmic compartments. Live HEK-293 cells expressing GFP-LXRbeta or beta -arrestin 2-GFP with or without ALK-1 Q-D as indicated were directly examined by confocal microscopy. A-C, the localization of GFP-LXRbeta in representative HEK-293 cells in the absence of ALK-1 Q-D. D-F, the localization of GFP-LXRbeta in representative HEK-293 cells in the presence of ALK-1 Q-D. Prominent membrane localization of GFP-LXRbeta in a representative cell is indicated by a white arrow in panel F. G, the localization of beta -arrestin 2-GFP in a representative HEK-293 cell. Data shown are representative of two independent experiments.

To investigate the potential physiological relevance of the interaction of ALK-1 and LXRbeta , we surveyed the expression patterns of ALK-1 and LXRbeta by performing virtual Northern blot analysis of SAGE and EST databases and by examining published Northern blot analysis of ALK-1 (28-30) and LXRbeta (31). These surveys confirmed that LXRbeta is widely expressed, whereas ALK-1 has a more limited pattern of expression, primarily in vascular tissues (Table I). Significantly, ALK-1 and LXRbeta were co-expressed in 94% of tissues, including in endothelial cells, where ALK-1 is specifically expressed.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Correlation of mRNA expression for ALK-1 and LXRbeta
Expression of mRNA in various tissues and cell lines for ALK-1 and LXRbeta as demonstrated by Northern blot analysis, the presence of expressed sequence tag (ESTs), or a serial analysis of gene expression (SAGE) tag in a library made from the source tissue. + indicates the presence of mRNA; - indicates the absence of mRNA. When relative levels are known through Northern blot analysis, these levels are shown with +++ indicating high levels of expression and ++ indicating lower levels of expression. Data were obtained from Refs. 28-31.

LXRbeta as a Substrate for the ALK-1 Kinase-- Because LXRbeta specifically interacts with activated ALK-1 (ALK-1 Q-D), we explored whether LXRbeta was a substrate for the ALK-1 serine/threonine kinase. HA-tagged LXRbeta was expressed in COS-7 cells in the presence and absence of either wild type, activated, or kinase-dead ALK-1. The cells were labeled with orthophosphate, and LXRbeta was immunoprecipitated with alpha HA antibody. Activated ALK-1 was able to specifically phosphorylate LXRbeta (Fig. 3A, lane 4), whereas ALK-1 (Fig. 3A, lane 3) and ALK-1 K-R (Fig. 3A, lane 5) were not. To characterize the phosphorylation of LXRbeta by activated ALK-1, LXRbeta phosphorylated in vivo by activated ALK-1 was subjected to phosphoamino acid analysis. In vivo phosphorylated LXRbeta was primarily phosphorylated on serine residues, with a trace amount of threonine phosphorylation and no detectable tyrosine phosphorylation (ratio of pSer:pThr:pTyr of 12:1:0), (Fig. 3B). To identify the number of phosphorylation sites, phosphopeptide mapping was performed. LXRbeta was phosphorylated in vivo on at least two distinct sites, with one major phosphopeptide (Fig. 3C).


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 3.   ALK-1 phosphorylates LXRbeta on more than one serine/threonine residue in vivo. A, COS-7 cells expressing ALK-1 or ALK-1 mutants and full-length HA-tagged LXRbeta as indicated were metabolically labeled with [32P]orthophosphate, lysed, and immunoprecipitated with alpha HA antibody. Immunoprecipitates were analyzed by SDS-PAGE and phosphoimager analysis (upper panel). The position of radiolabeled LXRbeta is indicated by an arrow. Portions of the total cell lysates (TL) were analyzed by Western blot to control for expression of the indicated proteins (lower panel). B, 32P-labeled LXRbeta (phosphorylated in vivo by ALK-1 Q-D) was subjected to phosphoamino acid analysis using one-dimensional thin layer electrophoresis and analyzed by phosphoimager analysis. The positions of co-electrophoresed unlabelled phosphoamino acids are indicated (pS, serine; pT, threonine, pY, tyrosine). C, tryptic digests of gel-purified in vivo 32P-labeled LXRbeta were resolved in two dimensions with electrophoresis in pH 1.9 buffer: pH 3.5 buffer (2:1) and chromatography in standard chromatography buffer and analyzed by phosphoimager analysis. The origin (Ori) is indicated by the +. Data shown are representative of two to three independent experiments.

ALK-1 has been proposed to form a signaling complex with the type II TGF-beta receptor (Tbeta RII) and endoglin in endothelial cells (11). To investigate the specificity of phosphorylation of LXRbeta by activated ALK-1, we investigated the ability of wild type ALK-1 to phosphorylate LXRbeta in the presence of Tbeta RII or Tbeta RII and endoglin in the presence and absence of TGF-beta 1. Although activated ALK-1 phosphorylated LXRbeta (Fig. 4A, lane 2), wild type ALK-1 could not (Fig. 4A, lane 1) even in the presence of Tbeta RII (Fig. 4A, lane 3), Tbeta RII and TGF-beta 1 (Fig. 4A, lane 4), or Tbeta RII, endoglin, and TGF-beta 1 (Fig. 4A, lane 5), suggesting that an uncharacterized ligand stimulates ALK-1 to phosphorylate LXRbeta in vivo. Because Tbeta RII was also HA-tagged (like LXRbeta ), autophosphorylated Tbeta RII was immunoprecipitated in these experiments as well (Fig. 4A, lanes 3-5), confirming that Tbeta RII was expressed and catalytically active. Tbeta RII also co-immunoprecipitated ALK-1 as has been previously reported (11), confirming expression of ALK-1 in these studies. Interestingly, consistent with a recent report (32), endoglin expression reduced autophosphorylation of Tbeta RII and also reduced phosphorylation of ALK-1 (Fig. 4A, compare lane 5 with lane 4).


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 4.   Activated ALK-1 specifically phosphorylates LXRbeta . A, COS-7 cells expressing ALK-1, ALK-1 Q-D, Tbeta RII, and/or endoglin as indicated along with full-length HA-tagged LXRbeta were treated with 200 pM TGF-beta 1 for 24 h as indicated, metabolically labeled with [32P]orthophosphate and subjected to immunoprecipitation with alpha HA antibody. Immunoprecipitates were analyzed by SDS-PAGE and phosphoimager analysis. The positions of LXRbeta , ALK-1, and Tbeta RII are indicated by arrows. B, COS-7 cells expressing ALK-1 Q-D, ALK-2 Q-D, or ALK-5 T-D as indicated along with full-length HA-tagged LXRbeta were metabolically labeled with [32P]orthophosphate and subjected to immunoprecipitation with alpha HA antibody. Immunoprecipitates were analyzed by SDS-PAGE and phosphoimager analysis. The position of LXRbeta is indicated by an arrow. C, COS-7 cells expressing ALK-1 Q-D as indicated along with full-length HA-tagged LXRbeta or HA-tagged LXRalpha were metabolically labeled with [32P]orthophosphate and subjected to immunoprecipitation with alpha HA antibody. Immunoprecipitates were analyzed by SDS-PAGE and phosphoimager analysis. The position of LXRbeta is indicated by an arrow. Portions of the total cell lysates (TL) were analyzed by Western blot to control for expression of the indicated proteins (lower panels). Data shown are representative of two to three independent experiments.

We then investigated the ability of other type I TGF-beta superfamily receptors to phosphorylate LXRbeta . Constitutively active ALK-2 (Fig. 4B, ALK-2 Q-D, lane 4) or constitutively active ALK-5 (Fig. 4B, ALK-5 T204D, lane 5) were unable to phosphorylate LXRbeta under the same expression conditions that resulted in phosphorylation of LXRbeta by activated ALK-1 (Fig. 4B, lane 3), confirming the specificity of this phosphorylation by activated ALK-1. Finally, to investigate the specificity of this phosphorylation we analyzed the ability of activated ALK-1 to phosphorylate LXRalpha in vivo. Activated ALK-1 was unable to phosphorylate LXRalpha in vivo (Fig. 4C, lane 3) under conditions it was able to phosphorylate LXRbeta (Fig. 4C, lane 5). The specific phosphorylation of LXRbeta (but not LXRalpha ) by ALK-1 in vivo strongly supports a physiologically relevant interaction between ALK-1 and LXRbeta .

Effect of LXRbeta on the ALK-1 Signaling Pathway-- Although the signaling pathway downstream of ALK-1 has not been elucidated, ALK-1 Q200D has been demonstrated to activate BMP-responsive promoters, including XVent2 (15). Therefore we assessed the ability of LXRbeta to affect ALK-1 Q200D-mediated Vent2 promoter-driven luciferase activity. ALK-1 Q200D was able to induce Vent2 promoter-driven luciferase activity (Fig. 5A), and LXRbeta was able to significantly reduce the ability of ALK-1 Q200D to induce Vent2 promoter-driven luciferase activity (Fig. 5A). To establish the specificity of this effect, we analyzed the effect of LXRbeta on ALK-1, ALK-2, or constitutively active ALK-2-mediated Vent2 promoter-driven luciferase activity. In addition to ALK-1 Q200D, only constitutively active ALK-2 was able to induce Vent2 promoter-driven luciferase activity, and LXRbeta was able to potently inhibit the ability of constitutively active ALK-2 to perform this function as well (Fig. 5B). In contrast, LXRbeta had little effect on the Vent2 promoter alone (Fig. 5A) or the PAI-1 based promoter, pE2.1, alone or in the presence of ALK-1 Q200D (Fig. 5C), suggesting that the effects of LXRbeta were specific. To further assess the specificity of LXRbeta effects on the Vent2 promoter, we examined the effects of LXRalpha . Under identical conditions, LXRalpha was unable to significantly affect either ALK-1 Q200D (Fig. 5D) or constitutively active ALK-2 (Fig. 5E)-mediated induction of Vent2 promoter-driven luciferase activity. Taken together, these results suggest that LXRbeta may be a specific modulator of ALK-1 and -2 signaling.


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 5.   LXRbeta specifically inhibits ALK-1 and -2 signaling. P19 cells (A, B, D, and E) or L6 (C) cells were transfected with 100 ng of the indicated ALK expression vectors, 100 ng of the indicated reporter genes, and 100 ng of LXRbeta or -alpha expression vectors. 48 h after transfection, the cells were lysed and the lysates assayed for luciferase activity. Data shown are from three experiments done in triplicate and are presented as the mean -fold stimulations (± S.E.) of the luciferase induced by constitutively active ALK-1 or -2 relative to mock-transfected (vector only) samples with * indicating p < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALK-1 and endoglin are two endothelial cell-specific receptors that are both essential for angiogenesis, and mutation of either receptor results in the human disease hereditary hemorrhagic telangiectasia (33). For these reasons there remains significant interest in the mechanisms by which these receptors function and/or signal. Earlier studies have established that both of these receptors bind TGF-beta ligand with identical isoform specificity (11, 34), both bind other TGF-beta receptors including Tbeta RII, and both form a complex with each other (35). Despite this knowledge, the signaling pathway downstream from these receptors has remained undefined. Several factors account for this. First, despite binding TGF-beta , neither ALK-1 nor endoglin has been shown to signal in a TGF-beta ligand-dependent fashion (28, 36). Second, both endoglin and ALK-1 can bind other ligands, with endoglin binding Activin A, BMP-2, and BMP-7 along with their respective ligand binding receptors (35) and ALK-1 binding Activin A along with the type II activin receptor as well as an uncharacterized ligand in serum (11, 28). Finally, when examined, effects of ALK-1 and endoglin have not been consistent with endoglin overexpression either decreasing (37) or increasing (32) TGF-beta signaling and constitutively activated ALK-1 resulting in Smad1, Smad5, and Smad8 phosphorylation (14, 15), activating BMP-responsive promoters including XVent2 (15) and TLX2 (14) and inhibiting the TGF-beta -responsive promoter, PAI-1 (11). Here we present evidence that the cytoplasmic domain of activated ALK-1 specifically binds the nuclear receptor LXRbeta as demonstrated by 1) the isolation of two distinct, nearly full-length clones of LXRbeta (but not LXRalpha ) in a yeast two-hybrid screen with the cytoplasmic domain of activated ALK-1 as a bait (Fig. 1A); 2) the specific interaction of the cytoplasmic domain of activated ALK-1 with LXRbeta in a yeast two-hybrid interaction assay (Fig. 1B); 3) the specific interaction of the cytoplasmic domain of ALK-1 with LXRbeta (but not LXRalpha ) in GST pull-down assays (Fig. 1C); and 4) the specific interaction of full-length endogenous ALK-1 with endogenous LXRbeta in co-immunoprecipitation studies (Fig. 1D). This interaction appears to be of physiological significance because 1) it results in the translocation of LXRbeta from the nuclear compartment to the cytoplasmic compartment (Fig. 2); 2) it results in the specific phosphorylation of LXRbeta (but not LXRalpha ) by ALK-1 (Figs. 3 and 4); and 3) LXRbeta (but not LXRalpha ) is able to specifically modulate a known cellular function of ALK-1, namely activation of XVent2 promoter-driven luciferase activity (Fig. 5). Taken together, these studies serve to establish LXRbeta as a potential mediator/modulator of ALK-1 signaling. Because the physiological ligand and signaling pathway downstream of ALK-1 has not been elucidated, we cannot determine the precise role that LXRbeta plays in the ALK-1 signaling pathway at this time. However, because LXRbeta is a transcription factor and has effects at the transcriptional level on at least one known target of ALK-1, LXRbeta may function by inhibiting the ALK-1 signaling pathway, either by directly inhibiting binding of other transcription factors or by recruiting transcriptional repressors. Studies are currently underway to evaluate these hypotheses.

ALK-2 is the type I receptor in the TGF-beta superfamily most closely related to ALK-1 and shares many properties with ALK-1. Similar to ALK-1, ALK-2 binds the type II activin receptor, binds Activin A along with the type II activin receptor, and can activate a PAI-1-based reporter gene when co-expressed with the type II activin receptor (28). However, Xenopus animal cap explant assays suggest that ALK-2 does not function as an activin receptor in vivo (38), and subsequent studies have suggested that ALK-2 acts as a BMP receptor. For example, ALK-2 can bind BMP-7 when expressed with the type II activin receptor (14). In addition, constitutively activated ALK-2, like ALK-1, can phosphorylate Smad1, Smad5, and Smad8 (14, 39) and can activate BMP-responsive promoters including Xvent2 (15) and TLX2 (14). Finally, ALK-2, like ALK-1, can complex with Tbeta RII and bind TGF-beta 1 along with Tbeta RII (40, 41). Thus, the current finding that the ALK-1 interacting protein, LXRbeta , can mediate ALK-1 signaling as well as ALK-2 signaling is not entirely unexpected. One substantial difference between ALK-1 and -2 is that, in contrast to ALK-1, ALK-2 is ubiquitously expressed (40), similar to LXRbeta (31), suggesting the LXRbeta may play a larger role in mediated/modulating ALK-2 signaling in vivo. We have demonstrated that ALK-2 does not bind LXRbeta (Fig. 1B) and activated ALK-2 does not significantly phosphorylate LXRbeta (Fig. 4B), suggesting that, at least in the case of ALK-2 signaling, phosphorylation of LXRbeta by ALK-2 may not be necessary for the effects of LXRbeta on ALK-2 signaling. The role and mechanism of action of LXRbeta in modulating ALK-2 signaling is currently under further investigation.

The results of our initial yeast two-hybrid screen with the cytoplasmic domain of activated ALK-1 were somewhat unexpected because they suggested an interaction with a nuclear receptor, LXRbeta , thought to be directly targeted to the nucleus. Our initial results supported an interaction in vivo, because ALK-1 could phosphorylate LXRbeta in vivo and LXRbeta could effect ALK-1 signaling in vivo. In addition, receptors in the TGF-beta superfamily have been demonstrated to internalize, and some studies have suggested that internalization may be essential for TGF-beta signaling (42, 43). Furthermore, in a least one report, a type I TGF-beta superfamily receptor, ALK-5, has been demonstrated to have a nuclear presence (44), and members of the nuclear receptor family have been demonstrated to have a presence in the cytoplasm (45). Thus, an interaction between LXRbeta and ALK-1 could conceivably occur in the nucleus or cytoplasm. Our findings that LXRbeta is expressed in both the nucleus and cytoplasm and that activated ALK-1 is able to induce translocation of LXRbeta from the nucleus and cytoplasm strongly support a physiological interaction between ALK-1 and LXRbeta and suggest that this interaction occurs in the cytoplasm.

LXRalpha and -beta were initially identified in low-stringency screens of human and rat liver cDNA libraries, respectively, (20, 46) as well as by degenerate PCR of cDNA from an osteoblastic cell line (16). LXRalpha and -beta are highly conserved isoforms with 77% identity in their DNA and ligand binding domains. Accordingly, both LXRalpha and -beta bind DNA as heterodimers of retenoid X receptor and preferentially bind to hormone response elements (LXREs) that consist of two hexanucleotide repeats (AGGTCA) separated by four nucleotides (DR-4) (46). In addition, both LXRalpha and -beta are activated by specific oxysterols including 24(S),25-epoxycholesterol, 22(R)-hydroxycholesterol, and 24(S)-hydroxycholesterol (21, 22). Despite these common properties, LXRalpha and -beta appear to have distinct functions in vivo. LXRalpha is expressed in a tissue-specific manner, primarily in tissues rich in lipid metabolism such as adipose tissue, the liver, kidney, and intestine, whereas LXRbeta is ubiquitously expressed (31). In addition, LXRalpha has been established as a sensor of dietary cholesterol (23), whereas LXRbeta knockout mice lack a metabolic/lipid metabolism phenotype (24). Finally, although all LXR target genes encode proteins that have major roles in controlling cholesterol and/or fatty acid metabolism, in every case LXRalpha either exclusively regulates their expression or has significantly higher activity than LXRbeta in activating these genes (23). These results highlight the need to define specific functions for LXRbeta . The present studies suggest that one of the functions of LXRbeta may be to regulate TGF-beta superfamily signaling, particularly for the ALK-1 and -2 receptors. Clarification of the role of LXRbeta in the ALK-1 pathway awaits further elucidation of this pathway.

Nuclear receptors are classically activated by binding their respective lipophilic ligands. However, most nuclear receptors are phosphorylated on serine and threonine residues, and their level of phosphorylation generally increases upon ligand binding (47). This phosphorylation has been demonstrated to regulate DNA binding, transcriptional activation, and/or the stability of these receptors. In addition, ligand-independent activation of nuclear receptors by phosphorylation has been reported (48). This has been best documented for the estrogen receptor (ER): (1) it can be activated by growth factors (epidermal and insulin-like growth factors) or activators of protein kinase A and protein kinase C; 2) ligand-independent activation is associated with altered phosphorylation of ER; and 3) mutation of mitogen-activated protein (MAP) kinase phosphorylation sites on ER is able to abrogate activation by epidermal growth factor (49). Kinases which have been demonstrated to phosphorylate nuclear receptors include cyclin-dependent kinases, MAP kinases, casein kinase II, protein kinase A, and protein kinase C (47). The current studies identify ALK-1 as a specific kinase for LXRbeta but not for LXRalpha . Because LXRalpha is not active in modulating ALK-1 signaling, these results suggest that this phosphorylation event is critical for regulating LXRbeta effects. Current studies are directed at identifying the site of phosphorylation of LXRbeta by ALK-1 and establishing the physiological relevance of this phosphorylation.

Oxysterols have well defined roles in regulating cholesterol, fatty acid, triglyceride, and sphingolipid metabolism (50). In addition, oxysterols have been implicated in regulating a diverse array of cellular processes, including calcium metabolism (51), mitochondrial function, apoptosis, and signal transduction (50). In terms of vascular biology, oxysterols have been implicated in the process of atherosclerosis (52) and have been demonstrated to induce apoptosis in endothelial cells (53) and vascular smooth muscle cells (54). Whether oxysterols, through their effects on LXRbeta , have effects on ALK-1 signaling in endothelial cells and vascular smooth muscle cells, both of which have a prominent role in angiogenesis and the pathogenesis of HHT, remains to be determined.

    ACKNOWLEDGEMENTS

We thank Dr. David Mangelsdorf for the LXRbeta , LXRalpha , and RXRalpha expression plasmids and Dr. Donald McDonnell for helpful discussions.

    FOOTNOTES

* This work was supported by American Cancer Society Grant ACS-IRG 83-006 (to G. C. B.) and NCI, National Institutes of Health Grants CA-91816 (to G. C. B.) and 5-T32-CA09307-24 (to J. M. and S. J. F.).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.

To whom correspondence should be addressed: Box 2631, Duke University Medical Center, Durham, NC 27710. Tel.: 919-668-1352; Fax: 919-668-2458; E-mail: blobe001@mc.duke.edu.

Published, JBC Papers in Press, October 18, 2002, DOI 10.1074/jbc.M210376200

    ABBREVIATIONS

The abbreviations used are: TGF, transforming growth factor; HHT, hereditary hemorrhagic telangiectasia; HA, hemagglutinin; GFP, green fluorescence protein; AF, activation factor; LXR, liver X receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve]
2. Guttmacher, A. E., Marchuk, D. A., and White, R. I., Jr. (1995) New Engl. J. Med. 333, 918-924[Free Full Text]
3. McAllister, K. A., Grogg, K. M., Johnson, D. W., Gallione, C. J., Baldwin, M. A., Jackson, C. E., Helmbold, E. A., Markel, D. S., McKinnon, W. C., Murrell, J., et al.. (1994) Nat. Genet. 8, 345-351[CrossRef][Medline] [Order article via Infotrieve]
4. Johnson, D. W., Berg, J. N., Baldwin, M. A., Gallione, C. J., Marondel, I., Yoon, S. J., Stenzel, T. T., Speer, M., Pericak-Vance, M. A., Diamond, A., Guttmacher, A. E., Jackson, C. E., Attisano, L., Kucherlapati, R., Porteous, M. E., and Marchuk, D. A. (1996) Nat. Genet. 13, 189-195[CrossRef][Medline] [Order article via Infotrieve]
5. Burrows, F. J., Derbyshire, E. J., Tazzari, P. L., Amlot, P., Gazdar, A. F., King, S. W., Letarte, M., Vitetta, E. S., and Thorpe, P. E. (1995) Clin. Cancer Res. 1, 1623-1634[Abstract]
6. Li, D. Y., Sorensen, L. K., Brooke, B. S., Urness, L. D., Davis, E. C., Taylor, D. G., Boak, B. B., and Wendel, D. P. (1999) Science 284, 1534-1537[Abstract/Free Full Text]
7. Bourdeau, A., Dumont, D. J., and Letarte, M. (1999) J. Clin. Invest. 104, 1343-1351[Medline] [Order article via Infotrieve]
8. Arthur, H. M., Ure, J., Smith, A. J., Renforth, G., Wilson, D. I., Torsney, E., Charlton, R., Parums, D. V., Jowett, T., Marchuk, D. A., Burn, J., and Diamond, A. G. (2000) Dev. Biol. 217, 42-53[CrossRef][Medline] [Order article via Infotrieve]
9. Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe, P. K., Li, L., Miyazono, K., ten Dijke, P., Kim, S., and Li, E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2626-2631[Abstract/Free Full Text]
10. Urness, L. D., Sorensen, L. K., and Li, D. Y. (2000) Nat. Genet. 26, 328-331[CrossRef][Medline] [Order article via Infotrieve]
11. Lux, A., Attisano, L., and Marchuk, D. A. (1999) J. Biol. Chem. 274, 9984-9992[Abstract/Free Full Text]
12. James, P., Halladay, J., and Craig, E. A. (1996) Genetics 144, 1425-1436[Abstract]
13. Wang, T., Donahoe, P. K., and Zervos, A. S. (1994) Science 265, 674-676[Abstract/Free Full Text]
14. Macias-Silva, M., Hoodless, P. A., Tang, S. J., Buchwald, M., and Wrana, J. L. (1998) J. Biol. Chem. 273, 25628-25636[Abstract/Free Full Text]
15. Chen, Y. G., and Massague, J. (1999) J. Biol. Chem. 274, 3672-3677[Abstract/Free Full Text]
16. Shinar, D. M., Endo, N., Rutledge, S. J., Vogel, R., Rodan, G. A., and Schmidt, A. (1994) Gene 147, 273-276[CrossRef][Medline] [Order article via Infotrieve]
17. Apfel, R., Benbrook, D., Lernhardt, E., Ortiz, M. A., Salbert, G., and Pfahl, M. (1994) Mol. Cell. Biol. 14, 7025-7035[Abstract/Free Full Text]
18. Song, C., Kokontis, J. M., Hiipakka, R. A., and Liao, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10809-10813[Abstract/Free Full Text]
19. Seol, W., Choi, H. S., and Moore, D. D. (1995) Mol. Endocrinol. 9, 72-85[Abstract/Free Full Text]
20. Teboul, M., Enmark, E., Li, Q., Wikstrom, A. C., Pelto-Huikko, M., and Gustafsson, J. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2096-2100[Abstract/Free Full Text]
21. Janowski, B. A., Willy, P. J., Devi, T. R., Falck, J. R., and Mangelsdorf, D. J. (1996) Nature 383, 728-731[CrossRef][Medline] [Order article via Infotrieve]
22. Lehmann, J. M., Kliewer, S. A., Moore, L. B., Smith-Oliver, T. A., Oliver, B. B., Su, J. L., Sundseth, S. S., Winegar, D. A., Blanchard, D. E., Spencer, T. A., and Willson, T. M. (1997) J. Biol. Chem. 272, 3137-3140[Abstract/Free Full Text]
23. Peet, D. J., Turley, S. D., Ma, W., Janowski, B. A., Lobaccaro, J. M., Hammer, R. E., and Mangelsdorf, D. J. (1998) Cell 93, 693-704[CrossRef][Medline] [Order article via Infotrieve]
24. Alberti, S., Schuster, G., Parini, P., Feltkamp, D., Diczfalusy, U., Rudling, M., Angelin, B., Bjorkhem, I., Pettersson, S., and Gustafsson, J. A. (2001) J. Clin. Invest. 107, 565-573[Medline] [Order article via Infotrieve]
25. Chang, C., Norris, J. D., Gron, H., Paige, L. A., Hamilton, P. T., Kenan, D. J., Fowlkes, D., and McDonnell, D. P. (1999) Mol. Cell. Biol. 19, 8226-8239[Abstract/Free Full Text]
26. Whitney, K. D., Watson, M. A., Goodwin, B., Galardi, C. M., Maglich, J. M., Wilson, J. G., Willson, T. M., Collins, J. L., and Kliewer, S. A. (2001) J. Biol. Chem. 276, 43509-43515[Abstract/Free Full Text]
27. Xiao, Z., Liu, X., Henis, Y. I., and Lodish, H. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7853-7858[Abstract/Free Full Text]
28. Attisano, L., Carcamo, J., Ventura, F., Weis, F. M., Massague, J., and Wrana, J. L. (1993) Cell 75, 671-680[CrossRef][Medline] [Order article via Infotrieve]
29. ten Dijke, P., Ichijo, H., Franzen, P., Schulz, P., Saras, J., Toyoshima, H., Heldin, C. H., and Miyazono, K. (1993) Oncogene 8, 2879-2887[Medline] [Order article via Infotrieve]
30. Panchenko, M. P., Williams, M. C., Brody, J. S., and Yu, Q. (1996) Am. J. Physiol. 270, L547-558[Medline] [Order article via Infotrieve]
31. Lu, T. T., Repa, J. J., and Mangelsdorf, D. J. (2001) J. Biol. Chem. 276, 37735-37738[Free Full Text]
32. Guerrero-Esteo, M., Sanchez-Elsner, T., Letamendia, A., and Bernabeu, C. (2002) J. Biol. Chem. 277, 29197-29209[Abstract/Free Full Text]
33. Blobe, G. C., Schiemann, W. P., and Lodish, H. F. (2000) N. Engl. J. Med. 342, 1350-1358[Free Full Text]
34. Cheifetz, S., Bellon, T., Cales, C., Vera, S., Bernabeu, C., Massague, J., and Letarte, M. (1992) J. Biol. Chem. 267, 19027-19030[Abstract/Free Full Text]
35. Barbara, N. P., Wrana, J. L., and Letarte, M. (1999) J. Biol. Chem. 274, 584-594[Abstract/Free Full Text]
36. ten Dijke, P., Yamashita, H., Ichijo, H., Franzen, P., Laiho, M., Miyazono, K., and Heldin, C. H. (1994) Science 264, 101-104[Abstract/Free Full Text]
37. Letamendia, A., Lastres, P., Botella, L. M., Raab, U., Langa, C., Velasco, B., Attisano, L., and Bernabeu, C. (1998) J. Biol. Chem. 273, 33011-33019[Abstract/Free Full Text]
38. Armes, N. A., and Smith, J. C. (1997) Development 124, 3797-3804[Abstract]
39. Chen, Y., Bhushan, A., and Vale, W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12938-12943[Abstract/Free Full Text]
40. Ebner, R., Chen, R. H., Shum, L., Lawler, S., Zioncheck, T. F., Lee, A., Lopez, A. R., and Derynck, R. (1993) Science 260, 1344-1348[Abstract/Free Full Text]
41. Ebner, R., Chen, R. H., Lawler, S., Zioncheck, T., and Derynck, R. (1993) Science 262, 900-902[Abstract/Free Full Text]
42. Zwaagstra, J. C., El-, Alfy, M., and O'Connor-McCourt, M. D. (2001) J. Biol. Chem. 276, 27237-27245[Abstract/Free Full Text]
43. Penheiter, S. G., Mitchell, H., Garamszegi, N., Edens, M., Dore, J. J., Jr., and Leof, E. B. (2002) Mol. Cell. Biol. 22, 4750-4759[Abstract/Free Full Text]
44. Zwaagstra, J. C., Guimond, A., and O'Connor-McCourt, M. D. (2000) Exp. Cell Res. 258, 121-134[CrossRef][Medline] [Order article via Infotrieve]
45. Lalli, E., Ohe, K., Hindelang, C., and Sassone-Corsi, P. (2000) Mol. Cell. Biol. 20, 4910-4921[Abstract/Free Full Text]
46. Willy, P. J., Umesono, K., Ong, E. S., Evans, R. M., Heyman, R. A., and Mangelsdorf, D. J. (1995) Genes Dev. 9, 1033-1045[Abstract/Free Full Text]
47. Weigel, N. L. (1996) Biochem. J. 319, 657-667[Medline] [Order article via Infotrieve]
48. Weigel, N. L., and Zhang, Y. (1998) J. Mol. Med. 76, 469-479[CrossRef][Medline] [Order article via Infotrieve]
49. Bunone, G., Briand, P. A., Miksicek, R. J., and Picard, D. (1996) EMBO J. 15, 2174-2183[Medline] [Order article via Infotrieve]
50. Schroepfer, G. J., Jr. (2000) Physiol. Rev. 80, 361-554[Abstract/Free Full Text]
51. Neyses, L., Locher, R., Stimpel, M., Streuli, R., and Vetter, W. (1985) Biochem. J. 227, 105-112[Medline] [Order article via Infotrieve]
52. Brown, A. J., and Jessup, W. (1999) Atherosclerosis 142, 1-28[CrossRef][Medline] [Order article via Infotrieve]
53. Lizard, G., Deckert, V., Dubrez, L., Moisant, M., Gambert, P., and Lagrost, L. (1996) Am. J. Pathol. 148, 1625-1638[Abstract]
54. Nishio, E., Arimura, S., and Watanabe, Y. (1996) Biochem. Biophys. Res. Commun. 223, 413-418[CrossRef][Medline] [Order article via Infotrieve]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
J. Henry-Berger, K. Mouzat, S. Baron, C. Bernabeu, G. Marceau, J.-P. Saru, V. Sapin, J.-M. A. Lobaccaro, and F. Caira
Endoglin (CD105) Expression Is Regulated by the Liver X Receptor Alpha (NR1H3) in Human Trophoblast Cell Line JAR
Biol Reprod, June 1, 2008; 78(6): 968 - 975.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
I. P. Torra, N. Ismaili, J. E. Feig, C.-F. Xu, C. Cavasotto, R. Pancratov, I. Rogatsky, T. A. Neubert, E. A. Fisher, and M. J. Garabedian
Phosphorylation of Liver X Receptor {alpha} Selectively Regulates Target Gene Expression in Macrophages
Mol. Cell. Biol., April 15, 2008; 28(8): 2626 - 2636.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
W. Wente, M. B. Brenner, H. Zitzer, J. Gromada, and A. M. Efanov
Activation of Liver X Receptors and Retinoid X Receptors Induces Growth Arrest and Apoptosis in Insulin-Secreting Cells
Endocrinology, April 1, 2007; 148(4): 1843 - 1849.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Gerin, V. W. Dolinsky, J. G. Shackman, R. T. Kennedy, S.-H. Chiang, C. F. Burant, K. R. Steffensen, J.-A. Gustafsson, and O. A. MacDougald
LXR{beta} Is Required for Adipocyte Growth, Glucose Homeostasis, and {beta} Cell Function
J. Biol. Chem., June 17, 2005; 280(24): 23024 - 23031.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Lux, C. Beil, M. Majety, S. Barron, C. J. Gallione, H.-M. Kuhn, J. N. Berg, P. Kioschis, D. A. Marchuk, and M. Hafner
Human Retroviral gag- and gag-pol-like Proteins Interact with the Transforming Growth Factor-{beta} Receptor Activin Receptor-like Kinase 1
J. Biol. Chem., March 4, 2005; 280(9): 8482 - 8493.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
K. R. Steffensen and J.-A. Gustafsson
Putative Metabolic Effects of the Liver X Receptor (LXR)
Diabetes, February 1, 2004; 53(90001): S36 - 42.
[Abstract] [Full Text]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
E. G. Lund, J. G. Menke, and C. P. Sparrow
Liver X Receptor Agonists as Potential Therapeutic Agents for Dyslipidemia and Atherosclerosis
Arterioscler. Thromb. Vasc. Biol., July 1, 2003; 23(7): 1169 - 1177.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/52/50788    most recent
M210376200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mo, J.
Right arrow Articles by Blobe, G. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mo, J.
Right arrow Articles by Blobe, G. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement