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Am J Physiol Heart Circ Physiol 285: H1675-H1683, 2003. First published June 5, 2003; doi:10.1152/ajpheart.00165.2003
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Identification of a CArG-independent region of the cysteine-rich protein 2 promoter that directs expression in the developing vasculature

Yung-Fu Chang,1,2 Jiao Wei,1 Xiaoli Liu,1 Yen-Hsu Chen,1,3 Matthew D. Layne,1 and Shaw-Fang Yet1

1Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and 2School of Biology and 3Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China

Submitted 19 February 2003 ; accepted in final form 2 June 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cysteine-rich protein (CRP)2 is a member of the LIM-only CRP family that is expressed in vascular smooth muscle cells (VSMC). To gain insight into the transcription of CSRP2 (gene name for CRP2) in VSMC, we analyzed the 5'-flanking sequence of the CSRP2 gene. We showed previously that 4,855 bp of the 5'-flanking sequence of the CSRP2 gene directed lacZ reporter gene expression, primarily in the VSMC of transgenic mice. To further define the regulatory sequences important for CSRP2 expression in VSMC, a series of promoter constructs containing deletions of the 5'-flanking sequence upstream of a nuclear-localized lacZ reporter gene were generated and analyzed. Similar to that observed in the –4855CSRP2-lacZ mice, {beta}-galactosidase reporter activity was detected in the developing great vessels, aorta, intersegmental arteries, umbilical vessels, endocardial cushions, and neural tube in the –3513-, –2663-, –795-, and –664CSRP2-lacZ lines. However, an internal deletion of bp –573 to –550 abolished the vascular, but not the neural tube, staining. Interestingly, no CArG box [CC(A/T)6GG] was present in the –795-bp fragment. Cotransfection experiments showed that dominant-negative serum response factor (SRF) did not repress CSRP2 promoter activity, which was different from the repressive effect of dominant-negative SRF on the SM22{alpha} promoter. Our data suggest the presence of a VSMC-specific element(s) within bp –573 to –550 of the CSRP2 5'-flanking sequence; however, in contrast to many other smooth muscle genes, transcriptional regulation of the CSRP2 gene is not dependent on SRF.

transgenic mice; blood vessels; serum response factor

MEMBERS OF THE LIM FAMILY of zinc-finger proteins determine cell lineage and regulate cell growth and differentiation (2, 9, 14, 41, 48–50, 54). Although the LIM domain is structurally similar to GATA-type zinc fingers, LIM domains alone do not bind to DNA (42). LIM domains may confer specific protein-protein interactions (1, 11, 51). The protein-protein interactions mediated by LIM domains are important in the assembly of multiprotein complexes, as in the actin cytoskeleton (4, 10, 51). The LIM-only cysteine-rich protein (CRP) family of LIM proteins is characterized by the presence of two tandem LIM domains, each followed by a short glycine-rich repeat (9, 19, 29, 53, 56). The CRP protein family includes CRP1, CRP2/SmLIM, and CRP3/MLP. CRP1 is expressed in most tissues and cell types, including vascular smooth muscle cells (VSMC) (17, 44, 55, 56). CRP2 is expressed in arterial smooth muscle cells (SMC) (19, 20), whereas CRP3 is expressed only in striated muscle cells (2). Members of the CRP family have been implicated in muscle differentiation in vertebrates. CRP3-null mice develop heart failure soon after birth (3), most likely because of a disruption of the cardiomyocyte cytoarchitecture. CRP1 interacts with the adhesion plaque protein zyxin (3, 48, 51) and with the actin cross-linking protein {alpha}-actinin (44). Zyxin and {alpha}-actinin are important regulators of the actin cytoskeleton (13, 43). Although the biological functions of CRP1 and CRP2 have not been elucidated completely, they may play similar roles in maintaining the cytoarchitecture of SMC and, thereby, affect SMC development and differentiation (29).

CRP2 differs from other SMC marker genes in that it is expressed in arterial, but not venous or visceral, SMC (19, 20). For example, smooth muscle (SM) {alpha}-actin, SM22{alpha}, and SM myosin heavy chain (MHC) are expressed in arterial, venous, and visceral SMC. CRP2 expression is downregulated in response to arterial wall injury, suggesting that CRP2 may play an important role in the dedifferentiation and proliferation of arterial SMC (19). This hypothesis is supported by a recent report that CRP2, together with GATA and serum response factor (SRF) transcription factors, facilitates the expression of SM marker genes in 10T1/2 cells (7). Therefore, understanding the transcriptional mechanisms regulating CRP2 gene expression may provide insight into the proliferation and phenotypic modulation of arterial SMC.

Investigations into the transcriptional control of several VSMC marker genes, including SM {alpha}-actin (30), SM22{alpha} (8, 21, 25, 37, 52), SM MHC (31, 33, 34), and calponin (35), have provided insights into the complex regulatory mechanisms of VSMC proliferation, differentiation, and phenotypic modulation (38–40, 46). These genes share a common cis-acting element, the CArG box [CC(A/T)6GG], which binds SRF and is necessary for their SMC-specific transcription. Understanding the transcriptional regulation of genes expressed in VSMC is further complicated by the different origins of VSMC during development (18, 22, 40). Cardiac neural crest cells contribute to the formation of VSMC in the great arteries, whereas lateral plate mesoderm contributes to the VSMC of systemic arteries and veins (18, 22, 40). Coronary artery SMC are derived from the proepicardial organ (18, 36).

We have previously cloned and characterized 4,855 bp of the mouse CSRP2 (gene name for CRP2) 5'-flanking sequence (57). This 4,855-bp fragment directed lacZ reporter gene expression in the outflow tract, dorsal aorta, carotid arteries, intersegmental arteries, and neural tube in embryonic day 11.5 mouse embryos (57). To advance our understanding of the complex regulatory mechanisms of VSMC-specific transcription, the focus of our present study was to further define the regulatory sequences important for VSMC expression of CRP2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Transgenic plasmid constructs. A series of CSRP2 promoter constructs containing deletions of the 5'-flanking sequence upstream of a nuclear-localized lacZ reporter were generated from the parental –4855CSRP2-lacZ construct (bp –4855 to +40) (57). The –3513CSRP2-lacZ plasmid was generated by subcloning the DrdI-Acc65I fragment (blunt-ended) from –4855CSRP2-lacZ into the SmaI site of pPD46.21 (12). The –2663CSRP2-lacZ plasmid was generated by subcloning the EcoRV-Acc65I fragment from –4855CSRP2-lacZ plasmid into the SmaI site of pPD46.21. The –4855CSRP2-lacZ plasmid was digested with BstEII and SalI, filled in with Klenow fragment of DNA polymerase I (New England BioLabs), and religated to generate the –795CSRP2-lacZ plasmid. To generate the –237CSRP2-lacZ plasmid, a SmaI fragment from –795CSRP2-lacZ was subcloned into the SmaI site of pPD46.21. To generate the –664CSRP2-lacZ construct, a PCR fragment spanning bp –664 to +40 of CSRP2 was subcloned into the SmaI site of pPD46.21. With the use of the –664CSRP2-lacZ construct as a template, site-directed mutagenesis was performed using Pfu polymerase to create an internal deletion of bp –573 to –550 to generate the –664{Delta}(–573/–550)CSRP2 transgenic construct. All constructs were confirmed by sequencing.

Luciferase reporter plasmid construct. To generate the –4855CSRP2-luc construct, the PstI (blunted) and KpnI CSRP2 promoter fragment containing bp –4855 to +40 was isolated from the –4855CSRP2-lacZ plasmid and cloned into the SmaI and KpnI sites of the luciferase reporter pGL2-Basic (Promega). To generate –3513-, –2663-, and –795CSRP2-luc constructs, we first isolated DrdI-KpnI, EcoRV-KpnI, and BstEII-KpnI fragments containing bp –3513 to +40, –2663 to +40, and –795 to +40, respectively, from the –4855CSRP2-lacZ plasmid. The 5' ends were then blunted and cloned into the SmaI and KpnI sites of pGL2-Basic. To generate –237CSRP2-luc, a SmaI fragment containing bp –237 to +40 of the CSRP2 promoter from –4855CSRP2-lacZ was cloned into the SmaI site of pGL2-Basic. All constructs were confirmed by sequencing.

Generation and analysis of transgenic mice. Constructs were digested with PstI and NotI to remove vector backbone, and the purified fragments were injected into the pronuclei of fertilized FVB mouse eggs (Brigham and Women's Hospital, Core Transgenic Mouse Facility). Transgenic mice and embryos harboring the CSRP2 promoter-lacZ reporter gene were identified by PCR and Southern blot analysis using genomic DNA prepared from tail biopsies or yolk sacs (23). Routine genotyping was performed with PCR using an upper primer from the CSRP2 promoter (bp –91 to –73, 5'-CGCACACCCCGAGGGGCAT-3') and a lower primer from the lacZ reporter gene (5'-CAGTTTGAGGGGACGACG-3') to amplify a 600-bp fragment spanning the junction between the CSRP2 promoter and the lacZ reporter gene. Two to seven independent transgenic founder lines were obtained from each construct. Transgenic embryos and tissues were fixed, stained for {beta}-galactosidase activity, and analyzed as described elsewhere (57). Animal protocols were approved by the Harvard Medical Area Standing Committee on Animals.

Cell culture and transient transfection assays. Rat aortic SMC (RASMC) and mouse aortic SMC (MASMC) were harvested from male Sprague-Dawley rats and embryonic day 18.5 mouse embryos, respectively, by enzymatic dissociation according to the method of Günther et al. (15) and cultured as described elsewhere (57). RASMC (passages 6–8) or MASMC (passages 6–7) were transfected by using FuGENE 6 reagents according to the manufacturer's instructions (Roche Molecular Biochemicals). Approximately 2 x 105 cells were plated onto each well of six-well plates, and the cells were allowed to attach overnight. To correct for differences in transfection efficiency, 500 ng each of the luciferase plasmids and pCMV{beta} (Clontech) were cotransfected into RASMC. For dominant-negative (DN) SRF experiments, luciferase plasmid –795CSRP2-luc or –441SM22{alpha}-luc (24), expression plasmids [pCGN expression vector plus various amounts of DN-SRF expression plasmid (pCGN-DN-SRF that contains amino acids 1–269) (24)], and pCMV{beta} were cotransfected into RASMC. Luciferase and {beta}-galactosidase activity was measured after 2 days (57). Each construct was transfected at least three times, and each transfection was performed in triplicate.

Electrophoretic mobility shift assays. Nuclear proteins were isolated from RASMC at passages 6–8 (47), and protein concentrations were determined using the Bio-Rad protein assay reagent. Complementary oligonucleotides from the CSRP2 promoter bp –574 to –549 (5'-CTGAAACCCGAAGCCTTTTGGCGCCA-3') and aortic carboxypeptidase-like protein (ACLP) promoter bp –157 to –119 (5'-AGTCTGGGCTCCGTGCTGCTCCGCCTCCCTCCCCCGCAG-3') (24) were annealed and end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase (New England Biolabs). DNA-binding assays were performed essentially as described elsewhere (45). To determine the specificity of the DNA-protein complexes for the CSRP2 probe, we performed competition assays with ~100-fold molar excess of unlabeled double-stranded oligonucleotides encoding the identical CSRP2 probe, a Sp1 consensus sequence (5'-ATTCGATCGGGGCGGGGCGAGC-3'), or an ACLP probe. To characterize the specific DNA-binding proteins, we incubated nuclear proteins with 2 µg of antibodies to Sp1 or Sp3 (Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
CSRP2-lacZ transgene expression in early developing embryos. We previously showed that 4855 bp of the mouse CSRP2 5'-flanking sequence directed lacZ reporter gene expression in the vasculature of embryonic day 11.5 transgenic embryos (57). Given that several smooth muscle gene promoters are active in skeletal or cardiac muscle during early embryonic development (27, 28, 37), we assessed {beta}-galactosidase activity of transgenic embryos at embryonic days 9.5 and 10.5 in two independent founder lines (n > 5 each line). Whole mount staining showed that at embryonic day 9.5 the lacZ transgene was expressed in the dorsal aorta, first branchial arch, and bulbus cordis of the heart (Fig. 1A). In contrast to the rostralmost somite expression of the CSRP1 promoter (28), the transgene was expressed in the caudalmost somites. By embryonic day 10.5, blue staining in the somites had disappeared (Fig. 1B) and the transgene was expressed in the dorsal aorta, first branchial arch, outflow tract of the heart, and neural tube (Fig. 1B).



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  		Fig. 1. Expression of the lacZ transgene during early mouse embryonic development. Results from whole mount staining of embryos from –4855CSRP2-lacZ transgenic lines are shown. Two independent lines had similar lacZ expression patterns except for slight differences in staining intensity. Representative embryos are shown. A: at embryonic day 9.5 (E9.5; n > 5 for each line), transgene expression was detected in dorsal aorta (DA), first branchial arch (BA), bulbus cordis of the heart tube (BC), and caudalmost somites (SO). B: at embryonic day 10.5 (E10.5; n > 5 for each line), transgene expression was detected in dorsal aorta, first branchial arch, outflow tract (OT) of the heart, and neural tube (NT). Blue staining was no longer present in the somites.

 

CSRP2 promoter region bp –795 to –237 is required for transgene expression in the vasculature. To further define the sequences important for CRP2 expression in VSMC in vivo, we generated a series of promoter constructs containing deletions of the 5'-flanking sequence, –3513-, –2663-, –795-, and –237CSRP2-lacZ, from the parental –4855CSRP2-lacZ construct. Independent transgenic founder lines were generated from each construct and analyzed for lacZ reporter gene expression.

Similar to –4855CSRP2 transgenic lines (57) (Fig. 2A), whole mount staining of a representative –3513CSRP2 transgenic embryonic day 11.5 embryo showed strong blue staining in the outflow tract, aorta, and neural tube (Fig. 2B). In addition, blue staining was present in the endocardial cushions (Fig. 2, F and G). Deletion of the 5'-flanking sequence to bp –2663 retained {beta}-galactosidase activity in the blood vessels (Fig. 2C). Transgene expression was detectable in the aorta, small vessels in the head, carotid arteries, intersegmental arteries branching from the aorta, and umbilical vessels (Fig. 2C). Outflow tract and endocardial cushions also showed strong staining (Fig. 2H). Additional deletion to bp –795 retained the expression pattern in the blood vessels (Fig. 2D). No staining was observed in the endocardial cushion (Fig. 2I), suggesting that the elements required for endocardial cushion expression are located between bp –2663 and –795. Further deletion to bp –237 abolished all {beta}-galactosidase activity (Fig. 2, E and J), indicating that the region between bp –795 and –237 of the CSRP2 promoter was required for transgene expression.



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  		Fig. 2. Region between bp –795 and –237 of the CSRP2 promoter is required for transgene expression in blood vessels of transgenic mice. Transgenic embryos (embryonic day 11.5) harboring 5' deletions of the CSRP2 promoter-lacZ reporter gene were isolated, fixed, and stained for {beta}-galactosidase activity (blue). A–E show results from whole mount staining of embryos: –4855CSRP2 (A; 2 founder lines, n > 12 for each line), –3513CSRP2 (B; 4 founder lines, n > 12 for each line), –2663CSRP2 (C; 2 founder lines, n > 6 for each line), –795CSRP2 (D; 4 founder lines, n > 12 for each line), and –237CSRP2 (E; 7 founder lines, n > 6 for each line) transgenic lines. F–J show higher magnification of outflow tract and endocardial cushion (ECC): –4855CSRP2 (F), –3513CSRP2 (G), –2663CSRP2 (H), –795CSRP2 (I), and –237CSRP2 (J) transgenic lines. AO, aorta; CA, carotid artery; IA, intersegmental artery; UV, umbilical vessel.

 

To determine whether the transgene was expressed in VSMC, we performed histological analysis of embryonic day 11.5 embryos stained for {beta}-galactosidase activity. Inasmuch as the –4855, –3513, –2663, and –795 lines had a similar expression pattern in the blood vessels, representative embryos are shown from the different transgenic lines. Analysis of histological sections from –2663CSRP2 embryonic day 11.5 transgenic embryos showed {beta}-galactosidase activity in the outflow tract and aorta (Fig. 3A). Sections of the embryos showed transgene expression in the medial layer of the paired dorsal aorta and neural tube (–4855CSRP2; Fig. 3B). Although we are not certain which cell types express CRP2 within the neural tube, Bermingham et al. (6) detected CRP2 expression in the sciatic nerve and cultured Schwann cells. Blue staining was evident in the endocardial cushions (–4855CSRP2; Fig. 3C). Furthermore, {beta}-galactosidase activity was observed in the arterial vessels, but not in the veins (–3513CSRP2; Fig. 3D). Higher magnification of the aorta revealed blue staining in VSMC, but not in endothelial cells (Fig. 3E). Expression of lacZ was also detected in the cardiac outflow tract (–795CSRP2; Fig. 3F).



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  		Fig. 3. Expression of lacZ transgene in vascular smooth muscle cells (VSMC) of blood vessels. Transgenic embryos (embryonic day 11.5) harboring 5' deletions of the CSRP2 promoter-lacZ reporter gene were isolated, fixed, and stained for {beta}-galactosidase activity (blue). A: lacZ staining in outflow tract, aorta, and small blood vessels (V) from a representative –2663CSRP2 embryo section. B: transgene expression in paired dorsal aorta and neural tube from –4855CSRP2 embryo sections counterstained with nuclear fast red. C: higher magnification of B showing blue staining of endocardial cushions. D: lacZ reporter gene activity detected in the aorta, but not in the veins (VE), from a –3513CSRP2 embryo. E: higher magnification of D showing transgene expression in smooth muscle cells (SMC) but not endothelial cells (EC) of blood vessels. F: outflow tract staining coming out of the heart from a –795CSRP2 embryo section.

 

Expression of the CSRP2 promoter in vasculature of transgenic mice before and after birth. To determine whether lacZ transgene expression in blood vessels persisted throughout later embryonic development and in postnatal periods, we analyzed the reporter gene activity in embryonic day 18.5 embryos and 2-wk-old mice. Similar lacZ expression patterns were observed in transgenic lines harboring the –4855-, –3513-, and –795CSRP2 deletions (Fig. 4). No staining was detected in –237CSRP2 transgenic lines (data not shown). Great vessels, including the aorta, pulmonary trunk, subclavian arteries, and carotid arteries, showed strong lacZ reporter gene activity (Fig. 4A). No {beta}-galactosidase activity was detected in the coronary vasculature (Fig. 4A). Positive staining for lacZ expression was also observed in the abdominal aorta and iliac arteries, but not in organs containing non-VSMC, such as the uterus or bladder (Fig. 4B). The faint diffuse blue staining in the kidney was not specific and was also observed in nontransgenic animals (data not shown). The great arteries in the outflow region of the heart, including the aortic arch, pulmonary trunk, and ductus arteriosus, which connects the pulmonary trunk and the descending aorta, were positively stained in transgenic lines harboring various lengths of the CSRP2 promoter, including deletion to bp –795, at embryonic day 18.5 (Fig. 4, C–E) and 2 wk after birth (Fig. 4, F–H). We performed histological analysis of embryonic day 18.5 embryos from –4855CSRP2-lacZ lines stained for {beta}-galactosidase activity. Blue staining was detected in the smooth muscle layers of thoracic (Fig. 5A) and abdominal aorta (Fig. 5B). Henderson et al. (16) showed by in situ analysis that CRP2 is expressed in the renal cortex tubules. No positive blue nuclear staining was visible in transgenic (Fig. 5C) or wild-type kidney sections (Fig. 5D), indicating that elements important for CRP2 expression in the kidney were not present between bp –4855 and +40 of the CSRP2 promoter.



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  		Fig. 4. Expression of the lacZ transgene in blood vessels of transgenic mice before and after birth. A: transgene expression in major arteries from a –4855CSRP2 embryonic day 18.5 (E18.5) embryo (n > 6). PT, pulmonary trunk; RCC and LCC, right and left common carotid arteries; RSC and LSC, right and left subclavian arteries; RA and LA, right and left atria; V, ventricles. B: blood vessel staining in abdominal region from a –4855CSRP2 E18.5 embryo (n > 6). BL, bladder; IL, iliac artery; KI, kidney; UT, uterus. C–H: lacZ reporter gene expression in the aortic arch (AA) and surrounding arteries in E18.5 embryos (C–E) and 2-wk-old mice (F–H) from –4855CSRP2 (C and F), –3513CSRP2 (D and G), and –795CSRP2 (E and H) transgenic lines. Dar, ductus arteriosus.

 


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  		Fig. 5. Histological examination of lacZ transgene expression in aorta and kidney. A: sections of thoracic aorta from a representative –4855CSRP2 E18.5 transgenic embryo (Fig. 4B) showing blue staining in smooth muscle layers. B: sections of abdominal aorta from the same –4855CSRP2 E18.5 transgenic embryo in A showing lacZ transgene expression in smooth muscle layers. C and D: blue staining was not present in cross sections of kidneys from the transgenic embryo in A (C) or wild-type E18.5 embryos (D).

 

CSRP2 promoter analysis in cultured VSMC. To analyze CSRP2 promoter activity in a more quantitative manner, we generated a series of promoter-luciferase constructs containing deletions of the 5'-flanking sequence, –4855, –3513, –2663, –795, and –237, corresponding to the analyzed transgenic lines. Cultured RASMC were transiently transfected with these constructs, and promoter activity was measured 2 days after transfection. The promoter constructs –4855-, –3513-, –2663-, and –795CSRP2-luc, which drove lacZ reporter gene expression in vivo, generated approximately five- to sevenfold higher luciferase activity in transfected RASMC than the –237 construct, which did not confer lacZ expression in transgenic mice (Fig. 6). Similar results were obtained when these constructs were transiently transfected into MASMC (data not shown). These data, together with the studies in transgenic mice, indicated that the region between bp –795 and –237 contains important elements for CSRP2 promoter activity and VSMC expression.



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  		Fig. 6. Deletion analysis of the CSRP2 promoter in rat arterial SMC. Fragments of the CSRP2 5'-flanking region corresponding to the respective transgenic lines were cloned into the luciferase reporter pGL2-Basic. These CSRP2 promoter-luciferase reporter plasmids (500 ng/well) were transiently transfected into rat aortic SMC (RASMC, 2 x 105 cells/well) in triplicate using FuGENE 6 transfection reagent. All wells received 500 ng of pCMV{beta} to normalize for transfection efficiency. After 48 h, cells were harvested for luciferase and {beta}-galactosidase activity assays. Luciferase activity is expressed relative to –237CSRP2-luc activity. Error bars, SE (n = 3–6).

 

SRF-independent transcriptional activity of the CSRP2 promoter. SRF binds to the CArG-box motif and mediates SMC-specific transcription of many smooth muscle differentiation marker genes, including SM22{alpha} (8, 21, 25, 37, 52) and SM {alpha}-actin (30). Interestingly, no CArG box was detected within the 795 bp of the 5'-flanking sequence of the CSRP2 gene. However, this fragment directed lacZ reporter gene expression in the vasculature of transgenic mice. To test the possibility that SRF may regulate CRP2 transcription in a DNA-independent fashion, we cotransfected the DN-SRF expression construct, which lacks the COOH-terminal transactivation domain, with –795CSRP2 or the SM22{alpha} promoter-luciferase reporter plasmid into RASMC. As reported previously (5, 24), DN-SRF repressed SM22{alpha} promoter activity in a dose-dependent manner (Fig. 7). In contrast, DN-SRF did not repress CSRP2 promoter activity (Fig. 7), suggesting that transcriptional regulation of the CSRP2 gene is not dependent on SRF.



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  		Fig. 7. CSRP2 promoter activity in RASMC is independent of serum response factor (SRF). RASMC were transiently transfected with –795CSRP2-luc (500 ng/well) and dominant-negative (DN) SRF or the corresponding empty vector (pCGN) to make the final amount of DNA per well identical; –441SM22{alpha}-luc was used as a control. All wells received 500 ng of pCMV{beta} to normalize for transfection efficiency. After 48 h, cells were harvested for luciferase and {beta}-galactosidase activity assays. Luciferase activity of each promoter construct without DN-SRF was set at 100%. Error bars, SE (n = 3–6).

 

CSRP2 bp –573 to –550 promoter sequence confers VSMC expression in vivo. The CArG box/SRF-independent transcription in VSMC was also observed in the ACLP promoter (24). The lacZ reporter gene expression in arterial and venous SMC (24) is driven by 2.5 kb of the 5'-flanking sequence of the ACLP gene that does not contain any CArG box. Interestingly, sequence comparison between bp –795 and –237 of the CSRP2 promoter and 2.5 kb of the ACLP promoter revealed an identical 13-bp fragment (5'-GAAACCCGAAGCC-3') corresponding to bp –572 to –560 and bp –80 to –68 of the CSRP2 and ACLP promoters, respectively. To determine the functional importance of this fragment in the CSRP2 promoter, we generated a –664CSRP2-lacZ and a corresponding transgenic construct that had a 24-bp deletion from bp –573 to –550, –664{Delta}(–573/–550)CSRP2-lacZ. We tested these constructs in vivo by examining the F0 embryos at embryonic day 11.5. Whole mount staining showed that 5' deletion to bp –664 retained the transgene expression pattern (Fig. 8A), similar to longer 5'-promoter sequences (Fig. 2). In contrast, internal deletion of bp –573 to –550 abolished {beta}-galactosidase activity in the vasculature, while the neural tube staining was retained (Fig. 8B). Histological sections showed blue staining in the aorta and neural tube of –664CSRP2-lacZ embryos (Fig. 8C). Blue staining was observed only in a small population of cells in the neural tube of –664{Delta}(–573/–550)CSRP2-lacZ embryos (Fig. 8D).



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  		Fig. 8. CSRP2 bp –573 to –550 promoter sequence confers VSMC expression in vivo. Purified DNA fragment containing CSRP2 promoter and lacZ reporter gene was injected into pronuclei of fertilized FVB mouse eggs, and F0 embryos at embryonic day 11.5 were analyzed for {beta}-galactosidase activity. A: 2 F0 embryos harbored the –664CSRP2-lacZ transgene and had a similar expression pattern. Transgene was expressed in aorta, outflow tract, carotid arteries, and neural tube. B:4F0 664{Delta}(–573/–550)CSRP2-lacZ embryos with a similar expression pattern. Transgene was detected in the neural tube, but not in blood vessels. C: transverse sections of F0 embryonic day 11.5 –664CSRP2-lacZ transgenic embryos showed blue staining in the aorta and neural tube. D: transverse sections of F0 embryonic day 11.5 –664{Delta}(–573/–550)CSRP2-lacZ transgenic embryos showed blue staining in the neural tube.

 

Nuclear proteins binding to the VSMC element(s) of the CSRP2 promoter. A previous study of the ACLP promoter indicated that GC-rich sequences (bp –157 to –119) that bind Sp1 may be important for SMC-selective expression (24). Thus we wanted to test whether the identified VSMC element(s) (bp –573 to –550) of the CSRP2 promoter binds Sp1 or Sp3 transcription factors. By using the DNA fragment encoding bp –574 to –549 as a probe in electrophoretic mobility shift assays, we detected a DNA-protein complex when the probe was incubated with nuclear extracts prepared from RASMC (Fig. 9). The complex was specific, because a 100-fold molar excess of unlabeled identical competitor abolished the binding complex. This DNA-protein complex was not competed away by consensus Sp1 oligonucleotides. Furthermore, this DNA-protein complex was not competed away by oligonucleotides encoding bp –157 to –119 of the ACLP promoter (24). In addition, Sp1 or Sp3 did not bind to CSRP2 VSMC elements, because Sp1 or Sp3 antibodies did not supershift the DNA-protein complex (Fig. 9). These results indicated the presence of nuclear protein(s) other than Sp1 and Sp3 in this complex. As a positive control, when the oligonucleotides encoding bp –157 to –119 of the ACLP promoter were used as a probe, two major complexes were identified in RASMC nuclear extracts (Fig. 9, I and II), as reported previously (24). In addition, Sp3 and Sp1 antibodies supershifted complexes I and II, respectively (Fig. 9).



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  		Fig. 9. Nuclear proteins binding to VSMC elements of CSRP2 promoter. Electrophoretic mobility shift assays were performed with double-stranded, 32P-labeled oligonucleotides corresponding to bp –574 to –549 of the CSRP2 promoter. Addition of nuclear extracts (10 µg) from RASMC to the CSRP2 probe resulted in a retarded DNA-protein complex (arrowhead on left). Complex was abolished by addition of identical unlabeled oligonucleotides (|) but not by addition of consensus Sp1 oligonucleotides or aortic carboxypeptidase-like protein (ACLP) oligonucleotides. Incubation of nuclear extracts with Sp1 or Sp3 antibody before the reaction did not shift the complex. As a positive control, gel mobility shift assays using 32P-labeled oligonucleotides corresponding to bp –157 to –119 of the ACLP promoter was performed. Addition of RASMC nuclear extracts (5 µg) to the ACLP probe resulted in 2 major complexes (I and II, arrows on right). Incubation of nuclear extracts with Sp1 or Sp3 antibody before the reaction produced supershifted bands (Sp1 and Sp3, arrows on right).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
To further elucidate the molecular mechanisms that control CRP2 expression in VSMC, we examined regulation of its promoter by deletion analysis in vivo. Analysis of CSRP2 promoter transgenic mice revealed that the 5'-flanking region between bp –573 and –550 is required for lacZ transgene expression in VSMC. In contrast to many SMC gene promoters, expression of the CSRP2 promoter in VSMC in vivo is independent of the CArG box and SRF.

Reporter gene expression of the CSRP2 promoter was restricted mainly to arteries and was not detected in veins or in tissues that contain nonvascular SMC, including the uterus and bladder. This observation is consistent with the observation that CRP2 is an arterial SMC protein (19, 20). Transgene expression was intense in the great arteries in the outflow region of the heart, but not in the coronary arteries. VSMC are derived from different embryonic origins (18, 22, 40). Cardiac neural crest cells contribute to the formation of SMC in the great arteries (18, 22, 40), whereas coronary SMC are derived from the proepicardial organ (18, 36). Our results suggest that the 5'-flanking CSRP2 promoter we examined was active in neural crest-derived SMC.

Expression patterns of the CSRP2 promoter-lacZ transgene differed from several SMC marker gene promoters. The lacZ transgene driven by SM {alpha}-actin and SM MHC promoters (including their first introns) is expressed in all SMC types, including visceral organs, coronary vasculature, and venous and arterial SMC (30, 32, 33). CArG-box motifs in their first introns appeared to control the SMC expression of these genes. The fragment containing 445 bp of SM22{alpha} 5'-proximal promoter, a well-characterized SMC gene promoter, directed lacZ reporter gene expression in arterial, but not visceral, SMC (27, 37), although endogenous SM22{alpha} is expressed in all SMC types (26). The arterial SMC expression of the SM22{alpha} promoter is similar to that of the CSRP2 promoter. In contrast to the CSRP2 promoter, it was demonstrated that the CArG box controls arterial SMC expression of SM22{alpha} (8, 52). Despite the similarity in the gene structure of CSRP2 (57) and CSRP1 (28), which is expressed in vascular and visceral SMC, >5 kb of CSRP1 5'-flanking sequence could not direct transgene expression in SMC. It was demonstrated that a CArG element within intron 1 of the CSRP1 gene is responsible for directing reporter gene expression in arterial, but not venous, SMC (28). It remains to be determined whether functional CArG boxes exist in intron 1 of the mouse CSRP2 gene.

These previous studies on SMC expression show that the cis-acting element CArG box and its cognate transcription factor SRF are essential in controlling SMC expression. In contrast to these SMC genes, the region between bp –795 and +40 of the CSRP2 proximal promoter that contains no CArG box was responsible for directing reporter gene expression in arterial SMC. Furthermore, our DN-SRF experiments indicated that transcriptional activity of the CSRP2 promoter was not dependent on CArG box-SRF interactions, nor does it require SRF in a DNA-independent fashion. The CArG box-SRF-independent transcription in VSMC was also observed in the ACLP promoter (24). Sequence comparison between CSRP2 and ACLP promoters revealed an identical 13-bp fragment corresponding to bp –572 to –560 and bp –80 to –68 of the CSRP2 and ACLP promoters, respectively. Transgenic embryos harboring –664CSRP2-lacZ (Fig. 8A) had the same expression pattern in the blood vessels as those harboring –4855CSRP2-lacZ (Fig. 2A). Interestingly, an internal deletion of 24 bp, including the 13-bp sequence, resulted in lacZ transgene expression in the neural tube, but not in the blood vessels, of mutant transgenic embryos harboring –664{Delta}(–573/–550)CSRP2-lacZ (Fig. 8B). These data indicate that bp –573 to –550 of the CSRP2 promoter confers VSMC-specific expression. Furthermore, our electrophoretic mobility shift analyses showed that Sp1 and Sp3 transcription factors did not bind to the identified 24-bp VSMC element(s) (Fig. 9). Comparison of this 24-bp sequence (conserved between mouse and rat and, partially, with human) with transcription factor databases (BCM Search Launcher, TRANSFAC) showed a similarity between the 3' region (5'-TTTGGCGC-3') and E2F transcription factor-binding sites. However, consensus E2F1 oligonucleotides (5'-ATTTAAGTTTCGCGCCCTTTCTCAA-3') did not compete away the binding complex in gel shift assays (data not shown).

In the present study, we have identified important CSRP2 promoter regulatory sequences for VSMC-specific expression. Furthermore, our results indicate that unique elements/mechanisms regulate expression of CRP2 in VSMC.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported in part by National Institutes of Health Grants HL-57977 (to S.-F. Yet) and AR-47861 (to M. D. Layne), American Heart Association Grant-in-Aid 0150329N (to S.-F. Yet), and the Harvard University-Kaohsiung Medical University alliance (to Y.-F. Chang and Y.-H. Chen).


   ACKNOWLEDGMENTS
 
We are grateful to the late Arthur Mu-En Lee for enthusiasm and support of our work. We thank Mark A. Perrella for helpful suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S.-F. Yet, Pulmonary Div., Brigham and Women's Hospital, 75 Francis St., Thorn 1333, Boston, MA 02115 (E-mail: syet{at}rics.bwh.harvard.edu).

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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