Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M511237200 on February 17, 2006

J. Biol. Chem., Vol. 281, Issue 16, 10849-10855, April 21, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/16/10849    most recent
M511237200v1
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 Chen, L.
Right arrow Articles by Wang, X. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, L.
Right arrow Articles by Wang, X. L.
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?

Human Prolyl-4-hydroxylase {alpha}(I) Transcription Is Mediated by Upstream Stimulatory Factors*

Li Chen{ddagger}§, Ying H. Shen{ddagger}§, Xinwen Wang{ddagger}§, Jing Wang{ddagger}§, Yehua Gan{ddagger}§, Nanyue Chen, Jian Wang{ddagger}§, Scott A. LeMaire{ddagger}§, Joseph S. Coselli{ddagger}§, and Xing Li Wang{ddagger}§1

From the {ddagger}Section of Adult Cardiothoracic Service, Texas Heart Institute at St. Luke's Episcopal Hospital and §Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030 and Department of Molecular Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030

Received for publication, October 16, 2005 , and in revised form, January 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolyl-4-hydroxylase {alpha}(I) (P4H{alpha}(I)) is the rate-limiting subunit for P4H enzyme activity, which is essential for procollagen hydroxylation and secretion. In the current study, we have characterized the human P4H{alpha}(I) promoter for transcription factors and DNA elements regulating P4H{alpha}(I) expression. Using a progressive deletion cloning approach, we have constructed pGL3-P4H{alpha}(I) recombinant plasmids. We have identified a positive regulatory region at the positions of bp –184 to –97 responsible for ~80% of the P4H{alpha}(I) promoter efficiency. Three E-boxes were located within this region, and the E-box at position bp –135 explains most of the regulatory capacity. Upstream stimulatory factors (USF1/USF2) were shown to bind on the E-box using chromatin immunoprecipitation assay. Suppression of USF1 and/or USF2 using specific short interference RNA resulted in a significant reduction in P4H{alpha}(I) promoter activity, and overexpressed USF1 or USF2 increased P4H{alpha}(I) promoter activity significantly. Although transforming growth factor beta1 increased the USF1/USF2-E-box binding and P4H{alpha}(I) promoter activity, this up-regulatory effect can be largely prevented by USF1/USF2-specific short interference RNA. On the other hand, cigarette smoking extracts, which have been shown to suppress P4H{alpha}(I) expression, inhibited the binding between the USF1/USF2 and E-box, resulting in a reduced P4H{alpha}(I) promoter activity. Furthermore, the E-box on the P4H{alpha}(I) promoter appeared to indiscriminately bind with either USF1 or USF2, with a similar outcome on the promoter efficiency. In conclusion, our study shows that USF1/USF2 plays a critical role in basal P4H{alpha}(I) expression, and both positive (transforming growth factor beta1) and negative (cigarette smoking extract) regulators appear to influence the USF-E-box interaction and affect P4H{alpha}(I) expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular matrix (ECM),2 the structural framework of all tissues, plays a key role in pathogenesis. In arterial wall, ECM holds various types of cells, including endothelial cells, vascular smooth muscle cells (VSMC), and fibroblasts, in their functional places. Dysregulated ECM is frequently associated with atherogenesis, vulnerable plaques, and aneurysm (1–5). ECM is maintained by a balanced synthesis and degradation of mainly collagens and elastins, which are in turn regulated by other molecules, e.g. fibrillin. Collagens form a multigene family with at least 39 members, the genes of which are known to be dispersed throughout at least 15 chromosomes. Collagen molecules consist of three identical or dissimilar polypeptide chains (called {alpha} chains) and contain at least one triple helical collagenous domain with repeating (GXY)n sequences, i.e. a glycine residue at every third amino acid, with the frequent presence of proline and 4-hydroxyproline in the X and Y positions, respectively. Most collagen molecules form supramolecular assemblies, and the superfamily can be divided into nine different families based on their polymeric structures or other features, such as collagens that form fibrils (types I–III, V, and XI). In normal arteries, fibrillar collagens I and III are the major constituents of the intima, media, and adventitia, whereas types I, III, IV, and V are in endothelial and VSMC basement membranes.

The biosynthesis of the precursors of fibrillar collagens (called procollagens) involves a number of post-translational modifications. The intracellular modifications require five specific enzymes, three collagen hydroxylases (6, 7) and two collagen glycosyltransferases (8, 9). Among these hydroxylases, prolyl 4-hydroxylase (P4H), a {alpha}2beta2 heterotetramer, plays a key role in collagen metabolism. The beta subunit is identical to the enzyme protein disulfide isomerase (6, 10) and is produced in excess of the {alpha} subunit, which contains a major portion of the catalytic sites of the enzyme, and the abundance of the {alpha} subunit restricts the enzymatic activity (6). P4H is an intracellular enzyme required for the synthesis and formation of all known types of collagen. It catalyzes the formation of hydroxyproline from proline residues located in repeating Xaa-Pro-Gly triplets in the procollagens during post-translational processing and is the process essential for folding the procollagen polypeptide chains into stable triple helical molecules (6). Inhibition of the P4H enzymes generates unstable collagen that is degraded inside the cell and is not secreted, which is associated with reduced collagen production (5). On the other hand, overexpression of the P4H{alpha}(I) gene is associated with excess collagen production (11). P4H isoenzymes are expressed in most tissues, although there may be tissue specificity with regard to type I or II isoforms for the {alpha} subunit (12–14). Although relatively little data is available for factors affecting P4H expression and activity, P4H may be regulated by nitric oxide, tumor necrosis factor {alpha}, transforming growth factor beta (TGFbeta), insulin-like growth factor 1, beta fibroblast growth factor, certain cytokines, and cigarette smoking (15–22). Environmental stimuli, such as hypoxia, can induce P4H{alpha}(I) expression at both transcriptional and post-transcriptional levels, which results in excess accumulation of collagen, a process relevant to fibrosis (23, 24).

The promoter of rat P4H{alpha}(I) has been described by Takahashi et al. (24). However, little is known with regard to the functional features of human P4H{alpha}(I) promoter (AL731563 [GenBank] ). In the present study, we characterized the elements in the promoter of human P4H{alpha}(I). We found that the E-box-like sequence (CACGGG), which has been shown to bind upstream stimulatory factors (USF) (25–27), in the position of bp –135 is vital for P4H{alpha}(I) transcription. Upstream stimulatory factor (USF1/USF2) appears to be the transcription factor that controls the basal P4H{alpha}(I) expression. Factors including TGFbeta1 and cigarette smoking extract (CSE) up-regulated and down-regulated the USF-E-box interaction and affected P4H{alpha}(I) expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—pGL3-basic (Promega, Madison WI) was used to construct pGL-P4H{alpha}(I) reporter vector to evaluate transcription efficiency. We used a PCR-based method to clone serially deleted P4H{alpha}(I) promoter fragments into the pGL3-basic vector at the KpnI and HindIII sites. Genomic DNA from HeLa cells was used as the template for the cloning. The inserts were confirmed for the correct sequence and orientation by direct sequencing. We named each recombinant pGL3 vector according to the size of the inserted P4H{alpha}(I) fragment (Table 1). The same antisense oligonucleotide primer that corresponded to the intron 1 region IVS1 + 76–IVS1 + 96 (5'-CCAAAGCTTACCACCACAGCGGGAAGGAAT-3') was used for the generation of all of the clones. A KpnI linker was incorporated into the 5'-end of the sense primers, and a HindIII linker was incorporated into the 5'-end of the antisense primers. The digested PCR products were subcloned into pGL3-basic at KpnI and HindIII sites. The mutated construct pGL-145M was obtained by site-directed mutagenesis with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions using primers specifically introducing a mutation at the E-box site of the P4H{alpha}(I) pGL-145 vector. The primers used for site-directed mutagenesis are as follows (underlines refer to mutated nucleotides): sense 5' AGC AGG GAG GCA CTC TCT TGG CGC CTG C-3; antisense 5'-GCA GGC GCC AAG AGA GTG CCT CCC TGC T-3'. The E-box mutation was confirmed by the direct sequencing. The USF1-pSG5 and USF2-pSG5 expression vectors were generous gifts from the laboratory of Dr. M. Sawadogo at the University of Texas MD Anderson Cancer Center, Houston, TX. Details of the vectors were described previously (28).


View this table:
[in this window]
[in a new window]
  		TABLE 1
Primers for the generation of serially deleted P4H{alpha}(I) promoter fragments

The same antisense oligonucleotide primer was used for the generation of all of the clones shown. A KpnI linker (CTGGTACC), shown in bold, was incorporated into the 5'-end of the sense primers, and a HindIII linker (CCAAAGCTT) was incorporated into the 5'-end of the antisense primers. The IVS1 indicates the location within the first intron counting from the end of exon 1 (Fig. 1A). The insertion sites of the cloned DNA fragments are at the multiple cloning region of the +5 (KpnI) and +53 (HindIII) upper stream of the luciferase gene.

 
Cell Culture, Transfection, and Luciferase Assays—HeLa cells purchased from the American Type Culture Collection (Masassas, VA) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C with 5% CO2. Human aortic VSMCs were purchased from Cambrex BioScience, Inc. (Walkersville, MD). VSMCs were cultured in smooth muscle basal medium containing 10% fetal bovine serum (Cambrex BioScience, Inc.), and the cells in passages 6–8 were used for the experiments. Plasmid DNA was prepared using a plasmid extraction kit (Qiagen Inc., Valencia, CA). One microgram of pGL-P4H{alpha}(I) hybrid DNA and/or 1 µg of siRNA or USF-pSG5 were transfected into the cells cultured in a 6-well plate with Lipofectamine 2000 (Invitrogen), and 0.25 µg of pSVbetagal (Promega), which expresses beta-galactosidase, was co-transfected to allow normalization of the transfection efficiency. After 4 h, the cells were treated with or without 10 ng/ml TGFbeta1 (Leinco Technologies, Inc., St. Louis, MO) for 24 h in the same medium but containing 0.2% fetal bovine serum. We also treated the transfected cells with CSE as described previously (29). Briefly, the smoke of a research cigarette (2R4F from Tobacco Health Research, University of Kentucky; nicotine, 0.85 mg/cigarette; tar, 9.70 mg/cigarette) was bubbled into a flask containing 20 ml of prewarmed F12K complete medium. The ignited cigarette was completely consumed in puffs by 10-ml syringe suction at one end of the tube within 5 min. The aqueous smoke extract was filtered through 0.2-µm syringe filter, and the pH of the CSE was adjusted to 7.4. The concentration of CSE was calculated in arbitrary units as cigarette equivalents/ml of the medium. CSE was used to treat the cells within 30 min of extraction. After 24 h, the cells were harvested and lysed in reporter lysis buffer (Promega), and cell lysates were assayed for luciferase and beta-galactosidase activities. Luciferase activity was measured with an FB12 luminometer (Berthold Detection Systems, Pforzheim, Germany) using luciferin (Promega) as the substrate. beta-Galactosidase was assayed in an assay buffer (Promega), and the activity was measured at 420 nm in a spectrophotometer. The luciferase activity, which reflects the promoter activity, was normalized to the beta-galactosidase to account for the variation in transfection efficiency.

For siRNA transfection, 100 nM siRNA was transfected into the cells cultured in a 6-well plate with Lipofectamine 2000. The siRNA duplex (Integrated DNA Technologies, Houston, TX) sequences for USF1 were: sense, 5'-GAC CCA ACC AGU GUG GCU ATT-3' and anti-sense, 5'-UAG CCA CAC UGG UUG GGU CTT-3'. The siRNA duplex sequences for USF2 were: sense, 5'-CCU CCA CUU GGA AAC GGU ATT-3', and antisense, 5'-UAC CGU UUC CAA GUG GAG GTT-3'. As the negative control siRNA, we used scrambled siRNA designed and tested by Ambion, Inc. (catalog number 4611G, Austin, Texas), which has no significant sequence similarities to mouse, rat, or human gene sequences.

Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was performed using the histone H3 ChIP assay kit according to the manufacturer's protocol (Upstate%20Biotechnology">Upstate Biotechnology, Charlottesville, VA). In brief, ~3 x 107 cells were used per ChIP assay. The cells were cross-linked with 1% formaldehyde at 37 °C for 10 min and rinsed twice with ice-cold phosphate-buffered saline. The cells were harvested by a brief centrifugation. Cell pellets were resuspended in SDS lysis buffer (50 mmol/liter Tris-HCl, pH 8.1, 10 mmol/liter EDTA, 1% SDS, and protease inhibitors). Sonication was performed on ice using a Sonifier II 450 (Branson, Danbury, CT) with a 3-mm tip set at duty cycle 20 and an output level 2 to achieve chromatin fragments ranging between 200 and 1000 bp in size, followed by centrifugation at 15,000 x g for 10 min at 4 °C. Supernatants were collected and diluted 10-fold in ChIP dilution buffer (a 20-µl aliquot was removed to serve as an input sample) followed with preimmunoprecipitation clearing with 80 µl of a mixture of salmon sperm DNA/protein A/protein G at 4 °C with rotation for 30 min. Immunoprecipitation was carried out with 2 µg of anti-USF1 or -USF2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight with rotation. After immunoprecipitation, 60 µl of a mixture of salmon sperm DNA/protein A/protein G was added and incubated at 4 °C with rotation for 30 min and followed by brief centrifugation. The precipitates were washed twice (5 min each at 4 °C) with low salt buffer, once with high salt buffer, and once with LiCl buffer. Then the precipitates were washed again with TE buffer (10 mM Tris-Cl 1 mM EDTA) twice for 5 min each. The immune complexes were extracted three times with 200 µl of elution buffer. The eluates and the input were heated at 65 °C for at least 4 h to reverse the cross-link by the addition of 20 µl of 5 mol/liter NaCl. After proteinase K treatment, DNA was extracted by phenol/chloroform solution and precipitated with ethanol. The recovered DNA was resuspended in 30 µl of H2O, and 4 µl was used for PCR. PCR was performed using sense (5'-AGC AGG GAG GCA CGG GCT TGG-3') and antisense (5'-TAC TTC CTA CCC TCA GCC CGC-3') primers. The PCR product (bp 183), which contained the USF binding E-box cis-element, was analyzed by 2% agarose gel electrophoresis. Anti-FLAG antibody (Sigma) served as the negative control in the assay.


Figure 1
View larger version (59K):
[in this window]
[in a new window]
  		FIGURE 1.
Structure of P4H{alpha}(I) transcriptional regulatory region. A, the nucleotide sequence of the promoter region of P4H{alpha}(I) is shown. The putative binding sites for transcriptional factors are underlined. The major transcriptional initiation site (solid circle) is designated +1. The starting positions for primers used in the serial cloning are indicated in the figure. B, schematic representation of the 5'-deleted P4H{alpha}(I) promoter-luciferase constructs and their transcriptional activities in HeLa cells. HeLa cells were transfected with the 5'-deletion P4H{alpha}(I) promoter-luciferase constructs. The relative luciferase activities were assessed. Results are expressed as the ratio of luciferase activity in each extract relative to that in the extract from HeLa cells transfected with pGL3-basic. The data were normalized to the activity of the co-transfected pSVbetagal plasmid and presented as the means ± S.D. of three independent experiments.

 
Real Time Quantitative Reverse Transcription (RT)-PCR—Primers were designed with Beacon Designer version 2.0 software. The primers for the human P4H{alpha}(I) mRNA were: sense, 5'-CGT CTC CAG GAT ACC TAC AA-3' and antisense, 5'-AAG CAG TCC TCA GCC GTT AG-3'. The primers for the housekeeping gene human beta-actin were: sense, 5'-CTG GAA CGG TGA AGG TGA CA-3' and antisense, 5'-AAG GGA CTT CCT GTA ACA ATG CA-3'. Total RNA was extracted from the cells with TRIzol (Invitrogen) according to the manufacturer's protocol. The RNA solution was treated with 1 unit of RNase-free DNase I (1 unit/µl; Promega) in a final volume of 20 µl at 37 °C for 15 min to remove trace amounts of genomic DNA contamination. The DNase I was inactivated by incubation at 75 °C for 10 min. One microgram of total RNA was used for reverse transcription. The mRNAs were reverse-transcribed into cDNAs with an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) containing a mixture of oligo(dT) and random primers. Real time PCR was performed using an iCycler iQ real time PCR detection system with a SYBR Green I Supermix kit (Bio-Rad) with the program running for 40 cycles at 95 °C for 20 s and 60 °C for 1 min. The PCR efficiency for the primers was examined by serially diluting the cDNA templates. The melting curve analysis was performed over the range 55–95 °C by monitoring SYBR Green fluorescence at increasing temperatures (0.5 °C increment changes at 10-s intervals). PCR-specific products were determined as a clear single peak, at the melting curves, of >80 °C. The specificity of primers was also confirmed as a single band with the correctly amplified fragment size through agarose gel electrophoresis of the real time RT-PCR products. Real time PCR was duplicated for each cDNA sample. Each gene mRNA level was acquired from the value of the threshold cycle (Ct) of the real time PCR as related to that of beta-actin through the formula 2{Delta}Ct ({Delta}Ct = beta-actin Ct–gene of interest Ct).


Figure 2
View larger version (8K):
[in this window]
[in a new window]
  		FIGURE 2.
Mutating the E-box reduces P4H{alpha}(I) promoter activity. Plasmids pGL-184, pGL-145, and the mutated construct pGL-145M were transfected into HeLa cells. The relative luciferase activities were assessed. Results are expressed as the ratio of luciferase activity in each extract relative to that in the extract from HeLa cells transfected with pGL3-basic. The data were normalized to the activity of the co-transfected pSVbetagal plasmid and presented as the means ± S.D. of three independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Positive Regulatory Element in the P4H{alpha}(I) 5'-Flanking Sequence—To clarify the regions responsible for the basal transcriptional regulation of the P4H{alpha}(I) gene, we prepared several reporter constructs by cloning the 5'-flanking sequence of the P4H{alpha}(I) gene (Fig. 1A) into the upstream of the luciferase reporter. We then transfected HeLa cells with these constructs containing the progressively deleted 5'-flanking regions of the P4H{alpha}(I) gene (Fig. 1B) and measured the luciferase activity of the resulting cell extracts. The deletion of the 5'-flanking sequence from bp –580 to –184 did not affect the transcription efficiency (Fig. 1B). However, a deletion up to bp –97 resulted in an ~80% decrease in the transcription activity. These results suggest that the basal transcription of the P4H{alpha}(I) gene was positively regulated by a region located between bp –184 and –97. One or more specific factors interacting with a cis-element(s) located between bp –184 and –97 may regulate the P4H{alpha}(I) promoter efficiency.

Effects of Mutation in the –135 E-box—Through a bioinformatics search, we have located three consensus E-box-like elements (CACGGG) located between bp –184 and –97. They are at bp –169, –158, and –135 (Fig. 1A). To confirm the importance of the E-boxes in P4H{alpha}(I) gene transcription, we generated a wild-type pGL-145 (Fig. 2) and a mutated reporter gene construct pGL-145M in which the –135 E-box-like sequence (CACGGG) was mutated to CACTCT. We then transfected HeLa cells with the pGL-184, pGL-145, and pGL-145M vectors (Fig. 2). The deletion of the 5'-flanking sequence from –184 to –145, which removed the –169 and –158 E-boxes, did not affect the transcription efficiency. The mutation of the –135 E-box (pGL-145M), however, resulted in an ~60% reduction in the activity. These results suggest that the basal transcription of the P4H{alpha}(I) gene was positively regulated by the –135 E-box.

Involvement of USF1/USF2 in the P4H{alpha}(I) Promoter Activity—The USF is one of the main families of transcription factors binding to the E-box (30, 31). To investigate the involvement of USFs in transactivating the P4H{alpha}(I) promoter, we first examined the effects of silencing USF1 and USF2 by siRNAs on the activity of the P4H{alpha}(I) promoter. Transfection of USF1 siRNA and/or USF2 siRNA into HeLa cells showed a significantly decreased pGL-145 promoter activity (Fig. 3A). Using HeLa cells, we further examined whether USF1/USF2 silencing could also suppress P4H{alpha}(I) expression. Indeed, the mRNA levels of the P4H{alpha}(I) gene measured by the quantitative real time RT-PCR were significantly reduced when USF1/USF2 were silenced (Fig. 3B). In the meantime, negative control siRNA had no effect on P4H{alpha}(I) expression. Our experiments further demonstrated that the degree of inhibition by the USF1 or USF2 siRNA appeared to be similar, and treatment with both siRNAs did not induce any additive effect comparable to the reduction by a single siRNA. These results suggest that the transcription of the P4H{alpha}(I) gene was positively regulated by the USF1/USF2, either one of which would be efficient for P4H{alpha}(I) expression.

We then overexpressed USF1 or USF2 in HeLa cells and human aortic VSMCs by co-transfecting pGL-145 vectors with USF1- or USF2-pSG5 vectors. As shown in Fig. 3C, USF1 and USF2 significantly up-regulated P4H{alpha}(I) promoter activities as measured by luciferase activity in VSMCs. The same effect was also observed in HeLa cells (data not shown). In addition, USF1 appeared to induce a higher P4H{alpha}(I) promoter activity than did USF2 (Fig. 3C). Such differential effect was not elucidated by the siRNA-mediated USF1 and/or USF2 suppression.

Direct Binding of USF1/USF2 to the P4H{alpha}(I) Promoter—To further assess the involvement of USF1/USF2 in P4H{alpha}(I) transcription, we examined whether USF transcription factors could bind to the E-box in the P4H{alpha}(I) promoter in vivo. The binding of E-box-binding proteins USF1/USF2 to the P4H{alpha}(I) promoter was illustrated by the ChIP assay (Fig. 3D). We then examined the effect of USF1/USF2 silencing on the binding activity of the USF1/USF2 using the ChIP assay. We found a decreased binding activity of both USF1 and USF2 to the E-box (Fig. 3D). Meanwhile, the negative control siRNA had a minimal effect on the binding.

Effects of TGFbeta1 on P4H{alpha}(I) Promoter Activity—P4H{alpha}(I) has a central role in the biosynthesis of collagens. TGFbeta1 can induce up-regulated collagen synthesis. To evaluate whether and how the P4H{alpha}(I) promoter would respond to TGFbeta1 stimulation, pGL-145 luciferase plasmids were transfected into HeLa cells. The normalized luciferase activity was calculated after TGFbeta1 treatment. We found a 2-fold increase in the luciferase activity after TGFbeta1 treatment (Fig. 4A). This effect was blocked by USF1/USF1 silencing using siRNAs (Fig. 4A), whereas negative siRNA had no effect. We further demonstrated the effect of TGFbeta1 treatment on the P4H{alpha}(I) mRNA levels in HeLa cells (Fig. 4B). We found a 1.8-fold increase in P4H{alpha}(I) mRNA levels, and this effect was blocked by USF1/USF2 silencing using siRNA (Fig. 4B). The same effect was also shown in VSMCs (data not shown). ChIP assay showed TGFbeta1 increased in the band intensity for the binding between the USF1/USF2 and the E-box in the P4H{alpha}(I) promoter (Fig. 4C). Furthermore, negative control siRNA had no effect on the binding activity. Taken together, these results suggest that the transcription of the P4H{alpha}(I) gene was positively regulated by TGFbeta1 through the USF1/USF2 and E-box interaction.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3.
Silencing of USF1/USF2 by short interference RNA (siRNA) decreased the activity of the P4H{alpha}(I) promoter and overexpressed USF1/USF2 increased the P4H{alpha}(I) promoter activity. A, 1 µg of pGL-145 hybrids was transfected with (+) or without (–) 100 nM USF1 siRNA and/or USF2 siRNA into HeLa cells cultured in a 6-well plate with Lipofectamine 2000. We used scrambled siRNA designed and tested by Ambion, which has no significant sequence similarities to mouse, rat, or human gene sequences, as the negative control siRNA. 0.25 µg of pSVbetagal was co-transfected to allow normalization of transfection efficiency. Results are expressed as the ratio of luciferase activity in each cell extract relative to that in the extract from cells transfected with pGL-145 but without siRNA treatment. B, HeLa cells were transfected with (+) or without (–) 100 nM USF1 siRNA and/or USF2 siRNA. After 24 h, the P4H{alpha}(I) mRNA level was quantitated by real time RT-PCR and normalized to the housekeeping gene beta-actin. Results are expressed as the ratio relative to HeLa cells without siRNA treatment. C, human aortic smooth muscle cells were co-transfected with 1 µg of pGL-145 hybrids and 1 µg of empty expression vector (empty pSG5) or expression constructs for USF1 or USF2 using Lipofectamine 2000. 0.25 µg of pSVbetagal was co-transfected to allow normalization of transfection efficiency. After 28 h, the cells were harvested for luciferase assay. Results in each extract are expressed as the ratio relative to that in the extract from human aortic smooth muscle cells co-transfected with pGL-145 and empty pSG5. D, ChIP assays were performed with anti-USF1 or anti-USF2 antibody for the HeLa cells treated with (+) or without (–) 100 nM USF1 siRNA or USF2 siRNA for 24 h. Primers for the P4H{alpha}(I) 5'-flanking region from –145 to +38 were used to amplify the DNA isolated from the ChIP assay. All data are presented as the means ± S.D. of three independent experiments. IP, immunoprecipitation.

 
Effects of CSE on P4H{alpha}(I) Promoter Activity—Previously, we have shown (15) that CSE could down-regulate P4H{alpha}(I) expression in endothelial cells and VSMCs. To evaluate the responsiveness of the P4H{alpha}(I) promoter to CSE treatment, pGL-145 luciferase plasmids were transfected into HeLa cells, and the normalized luciferase activity was calculated after CSE treatment. We found an ~50% reduction in the luciferase activity by the CSE treatment (Fig. 5A). We further showed that CSE also decreased the P4H{alpha}(I) mRNA levels in HeLa cells (Fig. 5B), which was consistent with our previous report (15) on endothelial cells and VSMCs. ChIP assay demonstrated that CSE decreased the band intensity for the binding between the USF1/USF2 transcription factor and the E-box in the P4H{alpha}(I) promoter (Fig. 5C). These results suggest that the transcription of the P4H{alpha}(I) gene was negatively regulated by CSE through USF1/USF2-E-box interaction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because collagen is the major structural protein in the ECM, which provides the framework for all tissues including arteries, there is little doubt that factors regulating collagen synthesis, process, secretion, and degradation will play a significant role in health and disease. It is therefore important to understand the molecular mechanisms regulating P4H{alpha}(I) expression, a rate-limiting subunit for the P4H enzyme, which is critical for procollagen modification and secretion (32, 33). Our study unequivocally shows that the E-box-like sequence located in the region of bp –184 to –97 upstream of the P4H{alpha}(I) promoter is the positive regulator and is responsible for nearly 80% of the basal promoter activity. As far as we are aware, this is the first characterization of the human P4H{alpha}(I) promoter (12, 34).

Although there is a significant sequence homology between human and rodent P4H{alpha}(I), the regulatory elements appear to be different between them. Takahashi and colleagues showed that the positive regulatory region is located at positions bp –246 to –165 in the rat P4H{alpha}(I) promoter (24). We showed that this positive regulator is located at bp –184 to –97, particularly the E-box at bp –135 in the human P4H{alpha}(I) promoter. Although their study did not show experimental evidence for any cis-acting element in P4H{alpha}(I) regulation, our experiments have shown convincing evidence for the involvement of the USF family and E-box interaction in regulating P4H{alpha}(I) expression. This conclusion is supported by the siRNA-specific suppression of the USF1/USF2, USF1/USF2 overexpression and the E-box mutagenesis. It should be noted, however, our study only demonstrates the regulatory elements for basal expression. DNA elements and corresponding transcription factors regulating the response to stimulation are likely to be different from those governing the basal expression.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4.
Effects of TGFbeta1 on the P4H{alpha}(I) promoter activity. A, one microgram of pGL-145 hybrids was transfected with (+) or without (–) 100 nM USF1 siRNA and/or USF2 siRNA into HeLa cells with Lipofectamine 2000. We used scrambled siRNA designed and tested by Ambion, which has no significant sequence similarities to mouse, rat, or human gene sequences, as the negative control siRNA. 0.25 µg of pSVbetagal was co-transfected to allow normalization of transfection efficiency. After 4 h, the cells were treated with or without 10 ng/ml TGFbeta1 in the same medium but containing 0.2% fetal bovine serum for 24 h. Results are expressed as the ratio of luciferase activity in each cell extract relative to that in the extract from cells transfected with pGL-145 but without TGFbeta1 and siRNA treatment. B, HeLa cells were transfected with or without 100 nM USF1 siRNA and/or USF2 siRNA and then treated with or without 10 ng/ml TGFbeta1 for 24 h. After 24 h, the P4H{alpha}(I) mRNA level was quantitated by real time RT-PCR and normalized to the housekeeping gene beta-actin. Results are expressed as the ratio relative to HeLa cells without TGFbeta1 and siRNA treatment. C, ChIP assays were performed with anti-USF1 or anti-USF2 antibody for HeLa cells transfected with or without 100 nM USF1 siRNA or USF2 siRNA and then treated with or without 10 ng/ml TGFbeta1 for 24 h. Primers for the P4H{alpha}(I) 5'-flanking region from bp –145 to +38 were used to amplify the DNA isolated from the ChIP assay. All data are presented as the means ± S.D. of three independent experiments. IP, immunoprecipitation.

 
USF1 and USF2 are ubiquitous proteins characterized by highly conserved C-terminal basic helix-loop-helix and leucine zipper domains responsible for dimerization and DNA binding activities (28, 35, 36). The E-box, the consensus sequence of which is CANNTG, is known as the binding site for USF and regulates the transcription of several genes (37, 38). They appear to be involved in the regulation of developmental and tissue-specific or gene-specific expression of a number of genes (39, 40), including those for surfactant protein A (41, 42), steroidogenic factor 1 (43), and carboxyl ester lipase (44). Our study adds a collagen regulator P4H{alpha}(I) as another USF target in regulating ECM metabolism in addition to its suppressing effect on cathepsin B expression (45). Together with a long list of genes including those involved in glucose metabolism, cholesterol metabolism, cell proliferation, detoxification, and hypoxia that can be potentially regulated by the USF (46–54), it appears clear that USF may have a role in vascular disease more than we have appreciated so far.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5.
Effect of CSE treatment on the P4H{alpha}(I) promoter activity. A, one microgram of pGL-145 hybrid DNA was transfected into HeLa cells with Lipofectamine 2000. 0.25 µg of pSVbetagal was co-transfected to allow normalization of transfection efficiency. After 4 h, the cells were then treated with or without CSE (0.01 unit) for 24 h. Results are expressed as the ratio of luciferase activity in each cell extract relative to that in the extract from cells transfected with pGL-145 but without CSE treatment. B, HeLa cells were treated with or without CSE (0.01 unit) for 24 h. After 24 h, the P4H{alpha}(I) mRNA level was quantitated by real time RT-PCR and normalized to the housekeeping gene beta-actin. Results are expressed as the ratio relative to HeLa cells without CSE treatment. C, ChIP assays were performed with anti-USF1 or anti-USF2 antibody for HeLa cells treated with or without CSE (0.01 unit) for 24 h. Primers for the P4H{alpha}(I) 5'-flanking region from bp –145 to +38 were used to amplify the DNA isolated from the ChIP assay. All data are presented as the means ± S.D. of three independent experiments. IP, immunoprecipitation.

 
The major USF species present in most tissues and cell lines is the heterodimer between USF1 and USF2, which interact with E-box (36). USF1 homodimers are less abundant, and USF2 homodimers are scarce (55, 56), which may have different effects (57, 58). In our study, however, we have shown that USF1 and USF2 appear to be interchangeable in P4H{alpha}(I) promoter activity. Suppressing either one will result in a similar degree of inhibition in P4H{alpha}(I) promoter activity; suppressing both does not induce an additive suppressing effect, which is consistent with the recent reports on TGFbeta1 (59, 60).

Our experiments have further shown that the positive effect of TGFbeta1 on collagen production is at least partly mediated by the up-regulated P4H{alpha}(I) expression, which is regulated through the USF-E-box interaction at the bp –135 of the P4H{alpha}(I) promoter. Likewise, the same promoter mechanism also appears to be negatively regulated by cigarette smoking, which has been shown to down-regulate P4H{alpha}(I) expression and collagen production (29). Clearly both TGFbeta1 and cigarette smoking would regulate ECM metabolism through more than one target. Our study shows the USF-E-box interaction on the P4H{alpha}(I) promoter is one of the targets. The same mechanism may apply to other target genes of TGFbeta1 and cigarette smoking if they have the same cis-acting element. However, we should be aware that the promoter elements responsible for basal expression may differ from those responding to stimulation. Although our experiments show that TGFbeta1 may regulate USF-E-box interaction, studies of others show that USF is also a positive transcription regulator for the TGFbeta1 gene via the glucose response element (59, 60). These findings suggest that, in addition to directly regulating P4H{alpha}(I) expression, USF proteins may regulate collagen or ECM metabolism through TGFbeta1 pathways. These effects could be cell type-specific. Together with their effects on growth factors, e.g. insulin-like growth factor-binding proteins (25), it appears that USF may have a significant role in ECM metabolism.

In summary, our study has characterized that USF plays an important role in P4H{alpha}(I) expression. The USF-E-box interaction appears to be the site wherein the positive (TGFbeta1) and negative (cigarette smoking) regulators of collagen production and P4H{alpha}(I) expression will act. Further studies on how TGFbeta1 or cigarette smoking regulates USF-E-box interaction will be of significant value in understanding vascular diseases, such as vulnerable atherosclerotic plaque or aortic aneurysm that involves dysregulated ECM metabolism.


   FOOTNOTES
 
* This work was supported by American Heart Association Established Investigator Award AHA 0440001N (to X. L. W.) and National Institutes of Health Grants HL066053 and HL71608. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: NAB 2010, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-5485; Fax: 713-798-1705; E-mail: xlwang{at}bcm.tmc.edu.

2 The abbreviations used are: ECM, extracellular matrix; P4H, prolyl-4-hydroxylase; USF, upstream stimulatory factor; ChIP, chromatin immunoprecipitation; siRNA, short interference RNA; VSMC, vascular smooth muscle cell; TGF, transforming growth factor; RT, reverse transcription; Ct, threshold cycle. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stary, H. C., Chandler, A. B., Glagov, S., Guyton, J. R., Insull, W., Jr., Rosenfeld, M. E., Schaffer, S. A., Schwartz, C. J., Wagner, W. D., and Wissler, R. W. (1994) Circulation 89, 2462–2478[Abstract/Free Full Text]
  2. Stary, H. C., Chandler, A. B., Dinsmore, R. E., Fuster, V., Glagov, S., Insull, W., Jr., Rosenfeld, M. E., Schwartz, C. J., Wagner, W. D., and Wissler, R. W. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 1512–1531[Abstract/Free Full Text]
  3. Fabunmi, R. P., Sukhova, G. K., Sugiyama, S., and Libby, P. (1998) Circ. Res. 83, 270–278[Abstract/Free Full Text]
  4. Libby, P. (1995) Circulation 91, 2844–2850[Free Full Text]
  5. Rocnik, E. F., Chan, B. M., and Pickering, J. G. (1998) J. Clin. Investig. 101, 1889–1898[Medline] [Order article via Infotrieve]
  6. Kivirikko, K. I., and Pihlajaniemi, T. (1998) Adv. Enzymol. Relat. Areas Mol. Biol. 72, 325–398[Medline] [Order article via Infotrieve]
  7. Kivirikko, K. I., and Myllyharju, J. (1998) Matrix Biol. 16, 357–368[CrossRef][Medline] [Order article via Infotrieve]
  8. Kivirikko, K. I., and Myllyla, R. (1982) Methods Enzymol. 82, 245–304[CrossRef][Medline] [Order article via Infotrieve]
  9. Prockop, D. J., and Kivirikko, K. I. (1995) Annu. Rev. Biochem. 64, 403–434[CrossRef][Medline] [Order article via Infotrieve]
  10. Kivirikko, K. I., Myllyla, R., and Pihlajaniemi, T. (1989) FASEB J. 3, 1609–1617[Abstract]
  11. John, D. C., Watson, R., Kind, A. J., Scott, A. R., Kadler, K. E., and Bulleid, N. J. (1999) Nat. Biotechnol. 17, 385–389[CrossRef][Medline] [Order article via Infotrieve]
  12. Annunen, P., Helaakoski, T., Myllyharju, J., Veijola, J., Pihlajaniemi, T., and Kivirikko, K. I. (1997) J. Biol. Chem. 272, 17342–17348[Abstract/Free Full Text]
  13. Annunen, P., Autio-Harmainen, H., and Kivirikko, K. I. (1998) J. Biol. Chem. 273, 5989–5992[Abstract/Free Full Text]
  14. Nissi, R., Autio-Harmainen, H., Marttila, P., Sormunen, R., and Kivirikko, K. I. (2001) J. Histochem. Cytochem. 49, 1143–1153[Abstract/Free Full Text]
  15. Raveendran, M., Senthil, D., Utama, B., Shen, Y., Dudley, D., Wang, J., Zhang, Y., and Wang, X. L. (2004) Biochem. Biophys. Res. Commun. 323, 592–598[CrossRef][Medline] [Order article via Infotrieve]
  16. Hiramatsu, M., Kumegawa, M., Hatakeyama, K., Yajima, T., Minami, N., and Kodama, H. (1982) Endocrinology 111, 1810–1816[Abstract/Free Full Text]
  17. Mizutani, K., Ikeda, K., and Yamori, Y. (2000) Biochem. Biophys. Res. Commun. 274, 61–67[CrossRef][Medline] [Order article via Infotrieve]
  18. Kato, Y., Inoue, H., Fujiyama, Y., and Bamba, T. (1996) J. Gastroenterol. 31, 565–571[CrossRef][Medline] [Order article via Infotrieve]
  19. Sasaki, T. (1992) J. Dermatol. 19, 664–666[Medline] [Order article via Infotrieve]
  20. Kawaguchi, Y., Kitani, A., Hara, M., Harigai, M., Hirose, T., Suzuki, K., Kawakami, M., Hidaka, T., Ishizuka, T., and Kawagoe, M. (1992) J. Rheumatol. 19, 1195–1201[Medline] [Order article via Infotrieve]
  21. Muguerza, B., Castilla-Cortazar, I., Garcia, M., Quiroga, J., Santidrian, S., and Prieto, J. (2001) Biochim. Biophys. Acta 1536, 185–195[Medline] [Order article via Infotrieve]
  22. Cao, M., Westerhausen-Larson, A., Niyibizi, C., Kavalkovich, K., Georgescu, H. I., Rizzo, C. F., Hebda, P. A., Stefanovic-Racic, M., and Evans, C. H. (1997) Biochem. J. 324, 305–310[Medline] [Order article via Infotrieve]
  23. Takahashi, Y., Takahashi, S., Shiga, Y., Yoshimi, T., and Miura, T. (2000) J. Biol. Chem. 275, 14139–14146[Abstract/Free Full Text]
  24. Takahashi, S., Dohi, N., Takahashi, Y., and Miura, T. (2002) Biochim. Biophys. Acta 1574, 354–358[Medline] [Order article via Infotrieve]
  25. Matsukawa, T., Inoue, Y., Oishi, Y., Kato, H., and Noguchi, T. (2001) Endocrinology 142, 4643–4651[Abstract/Free Full Text]
  26. Crissey, M. A., Leu, J. I., De Angelis, R. A., Greenbaum, L. E., Scearce, L. M., Kovalovich, K., and Taub, R. (1999) Hepatology 30, 1187–1197[CrossRef][Medline] [Order article via Infotrieve]
  27. Coulson, J. M., Edgson, J. L., Marshall-Jones, Z. V., Mulgrew, R., Quinn, J. P., and Woll, P. J. (2003) Biochem. J. 369, 549–561[CrossRef][Medline] [Order article via Infotrieve]
  28. Luo, X., and Sawadogo, M. (1996) Mol. Cell. Biol. 16, 1367–1375[Abstract]
  29. Raveendran, M., Wang, J., Senthil, D., Wang, J., Utama, B., Shen, Y., Dudley, D., Zhang, Y., and Wang, X. L. (2005) FEBS Lett. 579, 733–740[CrossRef][Medline] [Order article via Infotrieve]
  30. Kyriacou, C. P., and Rosato, E. (2000) J. Biol. Rhythms 15, 483–490[Free Full Text]
  31. Calomme, C., Nguyen, T. L., de Launoit, Y., Kiermer, V., Droogmans, L., Burny, A., and Van Lint, C. (2002) J. Biol. Chem. 277, 8775–8789[Abstract/Free Full Text]
  32. Kivirikko, K. I., Helaakoski, T., Tasanen, K., Vuori, K., Myllyla, R., Parkkonen, T., and Pihlajaniemi, T. (1990) Ann. N. Y. Acad. Sci. 580, 132–142[Medline] [Order article via Infotrieve]
  33. Myllyharju, J. (2003) Matrix Biol. 22, 15–24[CrossRef][Medline] [Order article via Infotrieve]
  34. Kukkola, L., Hieta, R., Kivirikko, K. I., and Myllyharju, J. (2003) J. Biol. Chem. 278, 47685–47693[Abstract/Free Full Text]
  35. Gregor, P. D., Sawadogo, M., and Roeder, R. G. (1990) Genes Dev. 4, 1730–1740[Abstract/Free Full Text]
  36. Sirito, M., Walker, S., Lin, Q., Kozlowski, M. T., Klein, W. H., and Sawadogo, M. (1992) Gene Expr. 2, 231–240[Medline] [Order article via Infotrieve]
  37. Jiang, B., and Mendelson, C. R. (2003) Mol. Cell. Biol. 23, 6117–6128[Abstract/Free Full Text]
  38. Jiang, B., and Mendelson, C. R. (2005) Mol. Cell. Biol. 25, 8824–8833[Abstract/Free Full Text]
  39. Qyang, Y., Luo, X., Lu, T., Ismail, P. M., Krylov, D., Vinson, C., and Sawadogo, M. (1999) Mol. Cell. Biol. 19, 1508–1517[Abstract/Free Full Text]
  40. Sawadogo, M. (1988) J. Biol. Chem. 263, 11994–12001[Abstract/Free Full Text]
  41. Gao, E., Wang, Y., Alcorn, J. L., and Mendelson, C. R. (1997) J. Biol. Chem. 272, 23398–23406[Abstract/Free Full Text]
  42. Gao, E., Wang, Y., Alcorn, J. L., and Mendelson, C. R. (2003) Am. J. Physiol. 284, L1027–L1036
  43. Harris, A. N., and Mellon, P. L. (1998) Mol. Endocrinol. 12, 714–726[Abstract/Free Full Text]
  44. Bengtsson, S. H., Madeyski-Bengtson, K., Nilsson, J., and Bjursell, G. (2002) Biochem. J. 365, 481–488[CrossRef][Medline] [Order article via Infotrieve]
  45. Yan, S., and Sloane, B. F. (2004) Gene 337, 199–206[CrossRef][Medline] [Order article via Infotrieve]
  46. Griffin, M. J., and Sul, H. S. (2004) IUBMB Life 56, 595–600[Medline] [Order article via Infotrieve]
  47. Shoulders, C. C., and Naoumova, R. P. (2004) Trends Mol. Med. 10, 362–365[CrossRef][Medline] [Order article via Infotrieve]
  48. Schweizer, M., Roder, K., Zhang, L., and Wolf, S. S. (2002) Biochem. Soc. Trans. 30, 1070–1072[CrossRef][Medline] [Order article via Infotrieve]
  49. Takahashi, Y., and Kamataki, T. (2001) Drug Metab. Rev. 33, 37–47[CrossRef][Medline] [Order article via Infotrieve]
  50. Girard, J., Ferre, P., and Foufelle, F. (1997) Annu. Rev. Nutr. 17, 325–352[CrossRef][Medline] [Order article via Infotrieve]
  51. Rousseau, G. G. (1992) Horm. Res. (Basel) 37, Suppl. 3, 88–92
  52. Sellak, H., Choi, C., Browner, N., and Lincoln, T. M. (2005) J. Biol. Chem. 280, 18425–18433[Abstract/Free Full Text]
  53. Pajukanta, P., Lilja, H. E., Sinsheimer, J. S., Cantor, R. M., Lusis, A. J., Gentile, M., Duan, X. J., Soro-Paavonen, A., Naukkarinen, J., Saarela, J., Laakso, M., Ehnholm, C., Taskinen, M. R., and Peltonen, L. (2004) Nat. Genet. 36, 371–376[CrossRef][Medline] [Order article via Infotrieve]
  54. Shoulders, C. C. (2004) Nat. Genet. 36, 322–323[CrossRef][Medline] [Order article via Infotrieve]
  55. Sirito, M., Lin, Q., Maity, T., and Sawadogo, M. (1994) Nucleic Acids Res. 22, 427–433[Abstract/Free Full Text]
  56. Sirito, M., Lin, Q., Deng, J. M., Behringer, R. R., and Sawadogo, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3758–3763[Abstract/Free Full Text]
  57. Rodriguez, C. I., Girones, N., and Fresno, M. (2003) J. Biol. Chem. 278, 43135–43145[Abstract/Free Full Text]
  58. Vallet, V. S., Casado, M., Henrion, A. A., Bucchini, D., Raymondjean, M., Kahn, A., and Vaulont, S. (1998) J. Biol. Chem. 273, 20175–20179[Abstract/Free Full Text]
  59. Weigert, C., Brodbeck, K., Sawadogo, M., Haring, H. U., and Schleicher, E. D. (2004) J. Biol. Chem. 279, 15908–15915[Abstract/Free Full Text]
  60. Zhu, Y., Casado, M., Vaulont, S., and Sharma, K. (2005) Diabetes 54, 1976–1984[Abstract/Free Full Text]

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
 Am. J. Physiol. Renal Physiol.Home page
L. Shi, D. Nikolic, S. Liu, H. Lu, and S. Wang
Activation of renal renin-angiotensin system in upstream stimulatory factor 2 transgenic mice
Am J Physiol Renal Physiol, February 1, 2009; 296(2): F257 - F265.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Liu, L. Shi, and S. Wang
Overexpression of upstream stimulatory factor 2 accelerates diabetic kidney injury
Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1727 - F1735.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. Zhang, M.-X. Zhang, Y. H. Shen, J. K. Burks, Y. Zhang, J. Wang, S. A. LeMaire, K. Yoshimura, H. Aoki, J. S. Coselli, et al.
TNF-{alpha} Suppresses Prolyl-4-Hydroxylase {alpha}1 Expression via the ASK1-JNK-NonO Pathway
Arterioscler. Thromb. Vasc. Biol., August 1, 2007; 27(8): 1760 - 1767.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
J.-i. Asaka, T. Terada, K. Ogasawara, T. Katsura, and K.-i. Inui
Characterization of the Basal Promoter Element of Human Organic Cation Transporter 2 Gene
J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 684 - 689.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/16/10849    most recent
M511237200v1
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 Chen, L.
Right arrow Articles by Wang, X. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, L.
Right arrow Articles by Wang, X. L.
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 © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement