179
Biochem. J. (1992) 286, 179-185 (Printed in Great Britain)
The transcriptional tissue specificity of the human proal(I) collagen gene is determined by a negative cis-regulatory element in the promoter Carl P. SIMKEVICH, James P. THOMPSON, Helen POPPLETON and Rajendra RAGHOW* Departments of Pharmacology and Medicine, University of Tennessee and the Veterans Affairs Medical Center, Memphis, TN 38104, U.S.A.
The transcriptional activity of plasmid pCOL-KT, in which human proal(I) collagen gene upstream sequences up to -804 and most of the first intron (+ 474 to + 1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human proal(I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of proal(I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to + 1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(- SstII) construct, which contained the intronic SstII fragment (+ 670 to + 1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5'-upstream sequences and the first intron determine the mesenchymal cell specificity of human proal(I) collagen gene transcription.
INTRODUCTION An important structural constituent of connective tissues, type I collagen is a major secretory gene product of mesenchymal cells (Miller & Gay, 1987; Kuhn, 1987; Vuorio & de Crombrugghe, 1990). Type I collagen is assembled as a heterotrimer consisting of two proal(I) and one proa2(I) chains; the two a chains are products of unique genes located on two separate chromosomes (Miller & Gay, 1987; Kuhn, 1987; Vuorio & de Crombrugghe, 1990). Collagen genes are controlled in both tissue- and
development-specific modes (Miller & Gay, 1987; Kuhn, 1987; Vuorio & de Crombrugghe, 1990), as well as in response to a number of biological and pharmacological stimuli (Raghow et al., 1984, 1986, 1987a; Roberts et al., 1986; Choe et al., 1987; Rossi et al., 1988; Penttinen et al., 1988; Postlethwaite et al., 1988; Solis-Herruzo et al., 1988; Cockyne & Cutroneo, 1988; Goldstein et al., 1990). Regulation of collagen genes in response to transforming growth factor , (Roberts et al., 1986; Raghow et al., 1987b; Rossi et al., 1988; Penttinen et al., 1988), fibrogenic factor (Raghow et al., 1984; Choe et al., 1987), interleukin 1 (Postlethwaite et al., 1988), tumour necrosis factor a (SolisHerruzo et al., 1988), phorbol esters (Goldstein et al., 1990) and glucocorticoids (Raghow et al., 1986; Cockyne & Cutroneo, 1988) has been demonstrated previously. Although both transcriptional and post-transcriptional mechanisms are involved
in the regulation of collagen gene expression in response to such diverse stimuli, the predominant mode of control under most conditions appears to be transcriptional (Raghow & Thompson, 1989; Bornstein & Sage, 1989; Ramirez & DiLiberto, 1990). A number of studies have shown that, in addition to the promoter-proximal sequences, DNA in the first intron modulates transcription of the human proal(I) collagen gene (Rossouw et al., 1987; Bornstein et al., 1987, 1988; Bornstein & McKay, 1988). However, the precise organization of sequence motifs in the promoter and the regulatory elements of the first intron of proa l(I) remains incompletely defined. Existence of both positive and negative regulatory sequences in the intron has been proposed. For instance, Bornstein and co-workers reported that a 274 bp (nucleotides + 820 to + 1093) AvaI fragment (A274) from the first intron of the human proal(I) collagen gene linked to a 430 bp fragment of the proal(I) promoter exhibited orientation-specific inhibition of the reporter gene (Bornstein et al., 1987). These authors also showed that nuclear extracts from HeLa, lymphoid and L cells recognized three sequence motifs, which correspond to bases 919-944, 951-978 and 986-1009 within the A274 fragment, suggesting that the trans-acting factors binding to A274 were present in both collagen-producing and non-producing cells. The complex array of additional regulatory sequences in the first intron was further suggested by the observation that the inhibitory effect of the A274 fragment could
Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CAT, chloramphenicol acetyltransferase. * To whom correspondence should be addressed: Veterans Affairs Medical Center, Research Service (151), 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A.
Vol. 286
180 be overridden by sequences surrounding it [e.g. 864 bp BglII/BamHI fragment (+ 544 to + 1407)]. Another important cis-regulatory sequence was identified recently by Liska et al. (1990), who showed that an Ap-l-like element was critically involved in the orientation-dependent enhancing ability of the intronic sequence. These investigators also provided evidence that the factor(s) which bind to the intronic Ap-1 site of the proal(I) gene were present only in the cells of mesenchymal origin (Liska et al., 1990). However, analyses of the Ap-l-like element of the human proacl (I) gene in our laboratory revealed results that differed from the data of Liska et al. (1990) in two important ways. First, the deletion of the Ap- 1-like element resulted in much greater expression of the plasmid pCOL-KT, suggesting that Ap-1 acted as a negative regulatory element. Second, the nuclear extracts from both collagen-producing and non-producing cells contained transacting factor(s) which were recognized by the Ap-l-like element (H. Katai, J. D. Stephenson, D. P. Simkevich, J. P. Thompson & R. Raghow, unpublished work). In another recent study, a minigene version of the human proal(I) gene containing 2.3 kb of the 5'-flanking sequence, with or without most of the first intron, was found to be expressed abundantly in stably transfected NIH 3T3 cells (Olsen et al., 1991). Although that study raised important questions about the contribution of intronic sequences in the regulation of the human proal(I) gene, it did not address the question of tissue specificity. Therefore, based on the published findings, we surmise that the questions regarding (1) the precise identity of determinants that confer tissue-specific expression to the human proal (I) gene, and (2) the contribution of the first intron to such transcriptional specificity, remain unanswered. A cursory comparison between the human and murine proal(I) collagen genes further complicates this situation, and suggests that there may be unique species-specific differences in the regulatory mechanisms of proal(I) gene expression (Rippe et al., 1989; Brenner et al., 1989). It was reported that, in contrast to what has been demonstrated for the human proal(I) gene, only 220 bp upstream of the transcription start site of the murine collagen proal (I) gene, containing two identical 12 bp repeats, were sufficient for maximal tissue-specific expression of the reporter gene. It is noteworthy, however, that even though the 12 bp sequences resembled the recognition sites of Sp-l and Ap-2 nuclear factors, the binding specificities of these sites were different from those of the authentic Sp-l and Ap-2 proteins (Rippe et al., 1989; Brenner et al., 1989). With the primary aim of defining the cis-acting sequences involved in tissue-specific transcriptional regulation, we created constructs in which expression of a reporter gene was driven by a sequence 2200 bp upstream of the transcription start site plus most of the first intron of the human proal(I) gene. Extensive deletion analysis of the promoter-proximal and the intronic sequences shows that: (i) 804 bp of upstream sequence was sufficient for optimal tissue-specific expression, and (ii) the determinants of mesenchymal cell specificity of proal() gene transcription consist of a negative regulatory element(s), removal of which leads to promiscuous expression of the proa I(I) gene in all cell types, regardless of their lineage. MATERIALS AND METHODS Materials Restriction endonucleases and other nucleic acid-modifying enzymes (purchased from Bethesda Research Laboratories, Gaithersberg, MD, U.S.A., and International Biotechnologies, New Haven, CT, U.S.A.) were used according to the
manufacturers' specifications. ['IC]-labelled chloramphenicol and acetyl-CoA were purchased from New England Nuclear,
C. P. Simkevich and others
Cambridge, MA, U.S.A., and Sigma, St. Louis, MO, U.S.A., respectively. Plasmids containing various DNA fragments were transfected into Escherichia coli strain JM 83, and bacteria were grown in LB broth under appropriate antibiotic selection conditions. Silica gel t.l.c. plates were obtained from J. T. Baker Inc., and were spray-coated with ENhance (New England Nuclear, Boston, MA, U.S.A.) and autoradiographed. Cells Continuous lines of human fibroblasts (HFL-1 and IMR-90), and a human erythroleukaemia cell line (K562) were obtained from American Tissue Culture Collection (ATCC). A rhabdomyosarcoma cell line (A204) was obtained from Dr. Helene Sage, University of Washington, Seattle, WA, U.S.A. Embryonic chicken fibroblasts (SL29) were purchased from ATCC, and a rat pheochromocytoma cell line (PC12) was obtained from Helen Fillmore, U.T. Memphis. HFL-1 and A204 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS) and antibiotics (100 units of penicillin/ml, 100,ug of streptomycin/ml and 25 of fungizone/ml; Gibco). K562 cells were grown in 50% DMEM/38 % RPMI/10 % FBS antibiotics/1 % glutamine. SL29 cells were cultivated in DMEM containing 10% FCS and antibiotics, and PC12 cells were grown in RPMI 1640 supplemented with 10 % FCS, 5% horse serum and antibiotics.
,g
Plasmid construction All recombinant DNA manipulations were carried out by standard protocols (Sambrook et al., 1989). The plasmid pCOLKT was constructed by isolating a 2.3 kb HindIll fragment of a genomic clone (Barsh et al., 1984) of human proal() and inserting it into the HindIll recognition sequence of the multiple cloning site of plasmid pKT (Reue et al., 1988). The translational initiation site and first exon (spanning nucleotides + 68 to + 478) of proal(I) were deleted as described (Thompson et al., 1991). Digestion of the 5 kb BamHI fragment of the genomic proal(I) DNA (Barsh et al., 1984) with SphI liberated a 1500 bp fragment representing additional 1197 bp of the 5'-upstream sequences. The SphI fragment was ligated into Sphl-digested pCOL-KT; recombinant clones with the correct orientation of the additional sequences, designated [pCOL-KT(L)], were grown and used for transfection studies.
Creation of deletion mutations Deletions of pCOL-KT were made using several different restriction endonucleases. pCOL-KT was digested with EcoRV to remove the fragment spanning nucleotides -625 to -442, and blunt-end-ligated with T4 DNA ligase to recircularize the plasmid. Similarly, digestion of pCOL-KT with XbaI deleted bases -804 to -331. Double digestions of pCOL-KT with XmaIII and SphI were used to remove bases -477 to -83; the ends were filled with Klenow enzyme and blunt-end-ligated. The putative regulatory sequences in the first intron were removed by digestion with SstII, which removed a fragment encompassing nucleotides + 670 to +1440, and recircularized. A number of double deletions were also constructed. pCOL-KT containing the EcoRV deletion was further digested with SstII to remove bases +670 to +1440, to create EcoRV/SstII (-625 to -442 and + 670 to +1440 deleted). Similarly, pCOL-KT containing the XbaI deletion was digested with SstII and recircularized to create XbaI/SstII (-804 to -331 and + 670 to + 1440 deleted). Partial digestion of pCOL-KT containing the SstII deletion with XbaI followed by relegation with T4 DNA ligase was done to create XbaI*/SstII (-804 to -609 and + 670 to +1440 deleted). Precise deletion points were confirmed by diagnostic enzyme fragmentation of the deleted constructs. We created XbaI* and
1992
Transcriptional control of proal(I) collagen gene XbaI*/(- SstII) by religating the SstII fragment in both orientations in the XbaI* construct after SstII digestion. Cell transfections and transient expression assays HFL- 1, A204 and SL29 cells were grown as monolayers to about 70 % confluency and transfected with pCOL-KT and its derivatives by the calcium phosphate precipitation method (Gorman et al., 1982); cells were glycerol-shocked for 2 min with 15 % (v/v) glycerol in Hanks balanced salt solution 4 h later (Corsaro & Pearson, 1981), washed three times with phosphatebuffered saline (138 mM-NaCl, 8.1 mM-NaH2PO4, 2 mmKH2PO4, 2.7 mM-KCI, 0.9 mM-CaCI2 and 0.5 mM-MgCI2) and incubated in fresh media at 37 °C for an additional 24-48 h. Before harvesting, cells were sequentially washed with cold phosphate-buffered saline and STE (0.04 M-Tris/Cl, pH 7.4, 1 mM-EDTA, 0.15 M-NaCl) and scraped; cell pellets were subjected to freeze-thaw cycles in 0.25 M-Tris/HCl, pH 7.8, the cell debris was pelleted and the cell extract was used for the chloramphenicol acetyltransferase (CAT) assay. Acetylation reactions were run for various lengths oftime for different cell types; reaction products were spotted on t.l.c. plates and developed in chloroform/methanol (19:1, v/v) (Gorman et al., 1982). The t.l.c. plates were sprayed with ENhance and exposed to XAR-2 Kodak film. The conversion of chloramphenicol to acetylated forms was quantified by two methods: autoradiographs were quantified by laser-assisted densitometry or by scraping the t.l.c. plates and measuring radioactivity by liquid scintillation spectroscopy. To correct for differences in transfection efficiencies, in most experiments the test constructs were co-transfected with a plasmid containing /,-galactosidase driven by the Rous sarcoma virus long-terminal repeat; fl-galactosidase activity was determined according to previously published procedures (Chang & Brenner, 1988). Additionally, to circumvent the intrinsic variation among different cell types, CAT assays were performed using constant amounts of total cellular protein, as determined by the Bradford assay (Bradford, 1976). RESULTS A collagen promoter/enhancer drives expression of a heterologous gene in a tissue-specific manner Plasmid pCOL-KT was generated by placing the 5'-noncoding and contiguous DNA spanning the first exon and intron of the proal(I) gene in front of the CAT gene, followed by removal of 410 bp (+ 68 to + 478) containing the first exon and the translation initiation site (Thompson et al., 1991). Conceivably, altered spatial relationships between the promoter and the first intron (due to a 410 bp deletion) in pCOL-KT, compared with the endogenous gene, could result in altered regulatory properties of such a construct. To circumvent the potential problem of artifactual expression of pCOL-KT, we had established earlier that the site of initiation of transcripts of endogenous proacl (I) and of pCOL-KT was identical (Thompson et al., 1991). Next, to test the transcriptional tissue specificity of the endogenous proal(I) gene promoter/enhancer, we transfected a variety of mesenchymal and non-mesenchymal cells and determined CAT activity by transient expression assays. A number of mesenchymal cell lines, including human fibroblasts (HFL- 1 and IMR-90) and a rhabdomyosarcoma cell line (A204), were transfected with pCOL-KT; parallel cultures were also transfected with pSV2CAT and pSVOCAT DNA to monitor expression of appropriate positive and negative controls. Expression of pCOL-KT occurred readily in HFL- I and A204 cells transfected with either pSV2CAT or pCOL-KT (Fig. 1); similar abundant expression of the reporter CAT gene was also seen in IMR-90 and NIH 3T3 cells and a number of human dermal and Vol. 286
181
synovial fibroblast lines (results not shown). Two cell lines of non-mesenchymal origin, a rat pheochromocytoma cell line (PC 12) and a human erythroleukaemia cell line (K562), which do not produce type I collagen, were also transfected with pCOLKT, pSVOCAT and pSV2CAT. While K562 and PC12 cells transfected with pSV2CAT, showed substantial exprepsion of CAT driven by the strong simian virus-40 promoter/enhancer, neither of these cells transfected with pCOL-KT expressed significant levels of CAT activity (Fig. 1). These data reveal that (i) the organization of the promoter-proximal and intronic regulatory sequences in pCOL-KT is compatible with the expression of appropriately initiated transcripts, and (ii) pCOL-KT expression occurs in a tissue-specific manner akin to the endogenous proal(I) gene. Although it seemed that sequence up to only 800 bp upstream of the transcription start site in pCOL-KT was sufficient for its expression in a tissue-specific manner, we were curious to investigate if other sequences further upstream were involved in the regulation of the proa I (I) gene. Thus we ligated an additional 1200 bp to pCOL-KT to create 'extended-promoter' pCOL-KT, [pCOL-KT(L)]. We found that although the patterns of tissuespecific expression of pCOL-KT(L) and pCOL-KT were identical, pCOL-KT(L) was expressed at significantly lower levels in a number of cell lines of mesenchymal origin, compared with pCOL-KT (H. Poppleton, J. Thompson & R. Raghow, unpublished work). Thus we elected to identify the cis-regulatory determinants of tissue specificity by site-specific alterations of pCOL-KT. A sequence located upstream of the proal(l) collagen gene promoter negatively regulates collagen gene expression The precise interactions among the transcriptional regulatory elements located in the 5'-upstream and intronic regions of the proal(I) gene remain somewhat unresolved and controversial. We reasoned that the two important sources of the discrepancies reported from different laboratories could be because either (i) (a)
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Fig. 1. The plasmid pCOL-KT is preferentially expressed in cells of mesenchymal lineage Two mesenchymal [HFL-1 (a) and A204 (b)] and two non-mesenchymal [K562 (c) and PC12 (d)] cell lines were transfected with pCOL-KT. Parallel cultures were not transfected with DNA (no DNA) or were transfected with a promoterless CAT plasmid (pSVOCAT) or with CAT driven by the simian virus 40 promoter/ enhancer (pSV2CAT). Cells were harvested 48 h after transfection and the cell extracts were assayed for conversion of '4C-labelled chloramphenicol into acetylated chloramphenicol by t.l.c. coupled with fluorography. pCOL-KT is readily expressed in HFL-1 and A204 cells and not expressed in K562 and PC12 cells. There is also abundant expression of pSV2CAT in all cell types regardless of their phenotype, while there is no expression of pSVOCAT in any of the cells.
C. P. Simkevich and others
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pCOL-KT
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determine the fraction of "C-labelled chloramphenicol into its acetylated derivatives. Single deletions to -442 (EcoRV), -804 to -331 (XbaI) or
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the previously tested constructs had artifactually spatial organization of the promoter and intronic leading to an altered mode of expression compared endogenous gene, or (ii) the intrinsic variations test systems (e.g. Kenopus oocytes versus support transcription of the proal(I) gene yielded observations. It should be emphasized, however, potential problems are not mutually exclusive, bination of the two could yield the variations different laboratories. To circumvent the first problem, pCOL-KT, in which the relationship between 5'-upstream sequences and the first intron was similar endogenous proal(I) gene. In the following wished to test the functional consequence of series double deletions in the proal(() promoter/enhancer step in our analysis of the cis-regulatory elements representative analysis of the relative expression or double deletion constructs is shown in Fig. from several independent determinations are summarized 3. In both HFL- I and A204 cells, the EcoRV (lacking -625 to -442) and Xbal (nt -804 to -331) changed
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of pCOL-KT showed significant decreases in CAT activity (2-10fold). Deletion of the SstII fragment in the intron (nt + 670 to + 1440) also led to a substantial decrease in expression compared with pCOL-KT. As anticipated, the XmaIII/SphI deletion mutant (nt -477 to -83) of pCOL-KT, which removed the canonical CAATT box located at -127, completely abolished expression of CAT (Fig. 2). Double deletion mutants of pCOL-KT were constructed by removing fragments of DNA after simultaneous digestion of the parent construct by two restriction enzymes (i.e. mixtures of EcoRV, XbaI and SstII), and the transcriptional activity of the deleted constructs was compared with that of pCOL-KT. Based on the representative data shown in Fig. 2, it appears that the negative impact of the single deletions in the promoter-proximal sequences was consistently relieved by deletion of the intronic sequences. For example, all three double deletion mutants (i.e. EcoV/SstII, XbaI/SstII and XbaI/SstII) were expressed at significantly greater levels compared with constructs containing only single deletions. Of all our constructs, the construct containing the XbaI*/SstII double deletion (lacking nucleotides -804 to -609 and + 670 to +1440) of pCOL-KT showed the most abundant expression: a 5-10-fold increase in CAT activity was observed in different experiments (results not shown). This remarkable effect of the XbaI*/SstII double deletions prompted us to test the effect of XbaI* deletion more extensively. We found that deletion with XbaI*, either alone or with the intronic SstII deletion, consistently boosted expression in all cell lines regardless of whether they were mesenchymal or non-mesenchymal in origin (see below).
that
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Determinants of tissue specificity of the transcriptional control of the human proal(l) gene are distributed in the promoterproximal and intronic sequences The phenotypes of the various deletion constructs of pCOLKT strongly support the previously published observations (Rossouw et al., 1987; Bornstein et al., 1987, 1988; Bornstein & McKay, 1988; Liska et al., 1990) showing that sequences located upstream of the start of transcription and in the first intron determine optimal expression of human proal (I) collagen. Our observations extend earlier results by demonstrating the existence of an additional upstream negative regulatory element in the proal(I) promoter (e.g. 'XbaI* domain'; nucleotides -804 to -609). We tested whether the XbaI* domain was involved in determining the tissue-specific expression of the human proclI(I) gene. A number of different cell lines of non-mesenchymal origin were transfected with pCOL-KT and its various deletion derivatives, and CAT activity was determined after transient expression. The representative data for two of these cell lines, K562 and PC12, are shown in Fig. 4. While pCOL-KT was not detectably expressed in either cell line, deletion with XbaI* (nt -804 to -609) resulted in excellent expression of CAT activity in both K562 and PC12 cells. Although introduction of the SstII deletion in XbaI* constructs to create XbaI*/SstII (nt -804 to -609 and + 670 to +1440) curtailed expression (5-10-fold), expression of the latter was significantly greater than that of pCOL-KT (Figs. 3 and 4). Interestingly, the other two constructs containing double deletions (e.g. EcoRV/SstII and XbaI/SstII) also showed slight expression in K562 cells (Figs. 3 and 4) and in PC12 cells (results not shown). To determine the contribution of the intronic sequences to the apparent breakdown of tissue specificity of transcription as a result of the XbaI* deletion, we reintroduced the intronic SstII fragment in both positive and negative orientations in the construct containing the XbaI*/SstII deletion. The XbaI* construct, which contains the +670 to
+ 1440 SstII intronic fragment in its natural orientation, expressed CAT to much greater extent than did any of the single
1992
Transcriptional control of proal(I) collagen gene
183 pCOL-KT
HindilI
SstlI
Sstll
+1
-804
+1440
+670
+68 to +478
Hindill +1447
pCOL-KT
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,
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.--fi,
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Relative CAT activity
HFL-1
A204
K562
PC-12
1.0
1.0
1.0
1.0
0.14±0.06 0.21±0.08
1.0
-
0.31 ± 0.04 0.21 ± 0.06
1.0
-
3.5 ± 0.21 2.9 ± 0.07
(-804 to -609)
Xmallil/Sphl
R t
Biochem. J. (1992) 286, 179-185 (Printed in Great Britain)
The transcriptional tissue specificity of the human proal(I) collagen gene is determined by a negative cis-regulatory element in the promoter Carl P. SIMKEVICH, James P. THOMPSON, Helen POPPLETON and Rajendra RAGHOW* Departments of Pharmacology and Medicine, University of Tennessee and the Veterans Affairs Medical Center, Memphis, TN 38104, U.S.A.
The transcriptional activity of plasmid pCOL-KT, in which human proal(I) collagen gene upstream sequences up to -804 and most of the first intron (+ 474 to + 1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human proal(I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of proal(I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to + 1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(- SstII) construct, which contained the intronic SstII fragment (+ 670 to + 1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5'-upstream sequences and the first intron determine the mesenchymal cell specificity of human proal(I) collagen gene transcription.
INTRODUCTION An important structural constituent of connective tissues, type I collagen is a major secretory gene product of mesenchymal cells (Miller & Gay, 1987; Kuhn, 1987; Vuorio & de Crombrugghe, 1990). Type I collagen is assembled as a heterotrimer consisting of two proal(I) and one proa2(I) chains; the two a chains are products of unique genes located on two separate chromosomes (Miller & Gay, 1987; Kuhn, 1987; Vuorio & de Crombrugghe, 1990). Collagen genes are controlled in both tissue- and
development-specific modes (Miller & Gay, 1987; Kuhn, 1987; Vuorio & de Crombrugghe, 1990), as well as in response to a number of biological and pharmacological stimuli (Raghow et al., 1984, 1986, 1987a; Roberts et al., 1986; Choe et al., 1987; Rossi et al., 1988; Penttinen et al., 1988; Postlethwaite et al., 1988; Solis-Herruzo et al., 1988; Cockyne & Cutroneo, 1988; Goldstein et al., 1990). Regulation of collagen genes in response to transforming growth factor , (Roberts et al., 1986; Raghow et al., 1987b; Rossi et al., 1988; Penttinen et al., 1988), fibrogenic factor (Raghow et al., 1984; Choe et al., 1987), interleukin 1 (Postlethwaite et al., 1988), tumour necrosis factor a (SolisHerruzo et al., 1988), phorbol esters (Goldstein et al., 1990) and glucocorticoids (Raghow et al., 1986; Cockyne & Cutroneo, 1988) has been demonstrated previously. Although both transcriptional and post-transcriptional mechanisms are involved
in the regulation of collagen gene expression in response to such diverse stimuli, the predominant mode of control under most conditions appears to be transcriptional (Raghow & Thompson, 1989; Bornstein & Sage, 1989; Ramirez & DiLiberto, 1990). A number of studies have shown that, in addition to the promoter-proximal sequences, DNA in the first intron modulates transcription of the human proal(I) collagen gene (Rossouw et al., 1987; Bornstein et al., 1987, 1988; Bornstein & McKay, 1988). However, the precise organization of sequence motifs in the promoter and the regulatory elements of the first intron of proa l(I) remains incompletely defined. Existence of both positive and negative regulatory sequences in the intron has been proposed. For instance, Bornstein and co-workers reported that a 274 bp (nucleotides + 820 to + 1093) AvaI fragment (A274) from the first intron of the human proal(I) collagen gene linked to a 430 bp fragment of the proal(I) promoter exhibited orientation-specific inhibition of the reporter gene (Bornstein et al., 1987). These authors also showed that nuclear extracts from HeLa, lymphoid and L cells recognized three sequence motifs, which correspond to bases 919-944, 951-978 and 986-1009 within the A274 fragment, suggesting that the trans-acting factors binding to A274 were present in both collagen-producing and non-producing cells. The complex array of additional regulatory sequences in the first intron was further suggested by the observation that the inhibitory effect of the A274 fragment could
Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CAT, chloramphenicol acetyltransferase. * To whom correspondence should be addressed: Veterans Affairs Medical Center, Research Service (151), 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A.
Vol. 286
180 be overridden by sequences surrounding it [e.g. 864 bp BglII/BamHI fragment (+ 544 to + 1407)]. Another important cis-regulatory sequence was identified recently by Liska et al. (1990), who showed that an Ap-l-like element was critically involved in the orientation-dependent enhancing ability of the intronic sequence. These investigators also provided evidence that the factor(s) which bind to the intronic Ap-1 site of the proal(I) gene were present only in the cells of mesenchymal origin (Liska et al., 1990). However, analyses of the Ap-l-like element of the human proacl (I) gene in our laboratory revealed results that differed from the data of Liska et al. (1990) in two important ways. First, the deletion of the Ap- 1-like element resulted in much greater expression of the plasmid pCOL-KT, suggesting that Ap-1 acted as a negative regulatory element. Second, the nuclear extracts from both collagen-producing and non-producing cells contained transacting factor(s) which were recognized by the Ap-l-like element (H. Katai, J. D. Stephenson, D. P. Simkevich, J. P. Thompson & R. Raghow, unpublished work). In another recent study, a minigene version of the human proal(I) gene containing 2.3 kb of the 5'-flanking sequence, with or without most of the first intron, was found to be expressed abundantly in stably transfected NIH 3T3 cells (Olsen et al., 1991). Although that study raised important questions about the contribution of intronic sequences in the regulation of the human proal(I) gene, it did not address the question of tissue specificity. Therefore, based on the published findings, we surmise that the questions regarding (1) the precise identity of determinants that confer tissue-specific expression to the human proal (I) gene, and (2) the contribution of the first intron to such transcriptional specificity, remain unanswered. A cursory comparison between the human and murine proal(I) collagen genes further complicates this situation, and suggests that there may be unique species-specific differences in the regulatory mechanisms of proal(I) gene expression (Rippe et al., 1989; Brenner et al., 1989). It was reported that, in contrast to what has been demonstrated for the human proal(I) gene, only 220 bp upstream of the transcription start site of the murine collagen proal (I) gene, containing two identical 12 bp repeats, were sufficient for maximal tissue-specific expression of the reporter gene. It is noteworthy, however, that even though the 12 bp sequences resembled the recognition sites of Sp-l and Ap-2 nuclear factors, the binding specificities of these sites were different from those of the authentic Sp-l and Ap-2 proteins (Rippe et al., 1989; Brenner et al., 1989). With the primary aim of defining the cis-acting sequences involved in tissue-specific transcriptional regulation, we created constructs in which expression of a reporter gene was driven by a sequence 2200 bp upstream of the transcription start site plus most of the first intron of the human proal(I) gene. Extensive deletion analysis of the promoter-proximal and the intronic sequences shows that: (i) 804 bp of upstream sequence was sufficient for optimal tissue-specific expression, and (ii) the determinants of mesenchymal cell specificity of proal() gene transcription consist of a negative regulatory element(s), removal of which leads to promiscuous expression of the proa I(I) gene in all cell types, regardless of their lineage. MATERIALS AND METHODS Materials Restriction endonucleases and other nucleic acid-modifying enzymes (purchased from Bethesda Research Laboratories, Gaithersberg, MD, U.S.A., and International Biotechnologies, New Haven, CT, U.S.A.) were used according to the
manufacturers' specifications. ['IC]-labelled chloramphenicol and acetyl-CoA were purchased from New England Nuclear,
C. P. Simkevich and others
Cambridge, MA, U.S.A., and Sigma, St. Louis, MO, U.S.A., respectively. Plasmids containing various DNA fragments were transfected into Escherichia coli strain JM 83, and bacteria were grown in LB broth under appropriate antibiotic selection conditions. Silica gel t.l.c. plates were obtained from J. T. Baker Inc., and were spray-coated with ENhance (New England Nuclear, Boston, MA, U.S.A.) and autoradiographed. Cells Continuous lines of human fibroblasts (HFL-1 and IMR-90), and a human erythroleukaemia cell line (K562) were obtained from American Tissue Culture Collection (ATCC). A rhabdomyosarcoma cell line (A204) was obtained from Dr. Helene Sage, University of Washington, Seattle, WA, U.S.A. Embryonic chicken fibroblasts (SL29) were purchased from ATCC, and a rat pheochromocytoma cell line (PC12) was obtained from Helen Fillmore, U.T. Memphis. HFL-1 and A204 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS) and antibiotics (100 units of penicillin/ml, 100,ug of streptomycin/ml and 25 of fungizone/ml; Gibco). K562 cells were grown in 50% DMEM/38 % RPMI/10 % FBS antibiotics/1 % glutamine. SL29 cells were cultivated in DMEM containing 10% FCS and antibiotics, and PC12 cells were grown in RPMI 1640 supplemented with 10 % FCS, 5% horse serum and antibiotics.
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Plasmid construction All recombinant DNA manipulations were carried out by standard protocols (Sambrook et al., 1989). The plasmid pCOLKT was constructed by isolating a 2.3 kb HindIll fragment of a genomic clone (Barsh et al., 1984) of human proal() and inserting it into the HindIll recognition sequence of the multiple cloning site of plasmid pKT (Reue et al., 1988). The translational initiation site and first exon (spanning nucleotides + 68 to + 478) of proal(I) were deleted as described (Thompson et al., 1991). Digestion of the 5 kb BamHI fragment of the genomic proal(I) DNA (Barsh et al., 1984) with SphI liberated a 1500 bp fragment representing additional 1197 bp of the 5'-upstream sequences. The SphI fragment was ligated into Sphl-digested pCOL-KT; recombinant clones with the correct orientation of the additional sequences, designated [pCOL-KT(L)], were grown and used for transfection studies.
Creation of deletion mutations Deletions of pCOL-KT were made using several different restriction endonucleases. pCOL-KT was digested with EcoRV to remove the fragment spanning nucleotides -625 to -442, and blunt-end-ligated with T4 DNA ligase to recircularize the plasmid. Similarly, digestion of pCOL-KT with XbaI deleted bases -804 to -331. Double digestions of pCOL-KT with XmaIII and SphI were used to remove bases -477 to -83; the ends were filled with Klenow enzyme and blunt-end-ligated. The putative regulatory sequences in the first intron were removed by digestion with SstII, which removed a fragment encompassing nucleotides + 670 to +1440, and recircularized. A number of double deletions were also constructed. pCOL-KT containing the EcoRV deletion was further digested with SstII to remove bases +670 to +1440, to create EcoRV/SstII (-625 to -442 and + 670 to +1440 deleted). Similarly, pCOL-KT containing the XbaI deletion was digested with SstII and recircularized to create XbaI/SstII (-804 to -331 and + 670 to + 1440 deleted). Partial digestion of pCOL-KT containing the SstII deletion with XbaI followed by relegation with T4 DNA ligase was done to create XbaI*/SstII (-804 to -609 and + 670 to +1440 deleted). Precise deletion points were confirmed by diagnostic enzyme fragmentation of the deleted constructs. We created XbaI* and
1992
Transcriptional control of proal(I) collagen gene XbaI*/(- SstII) by religating the SstII fragment in both orientations in the XbaI* construct after SstII digestion. Cell transfections and transient expression assays HFL- 1, A204 and SL29 cells were grown as monolayers to about 70 % confluency and transfected with pCOL-KT and its derivatives by the calcium phosphate precipitation method (Gorman et al., 1982); cells were glycerol-shocked for 2 min with 15 % (v/v) glycerol in Hanks balanced salt solution 4 h later (Corsaro & Pearson, 1981), washed three times with phosphatebuffered saline (138 mM-NaCl, 8.1 mM-NaH2PO4, 2 mmKH2PO4, 2.7 mM-KCI, 0.9 mM-CaCI2 and 0.5 mM-MgCI2) and incubated in fresh media at 37 °C for an additional 24-48 h. Before harvesting, cells were sequentially washed with cold phosphate-buffered saline and STE (0.04 M-Tris/Cl, pH 7.4, 1 mM-EDTA, 0.15 M-NaCl) and scraped; cell pellets were subjected to freeze-thaw cycles in 0.25 M-Tris/HCl, pH 7.8, the cell debris was pelleted and the cell extract was used for the chloramphenicol acetyltransferase (CAT) assay. Acetylation reactions were run for various lengths oftime for different cell types; reaction products were spotted on t.l.c. plates and developed in chloroform/methanol (19:1, v/v) (Gorman et al., 1982). The t.l.c. plates were sprayed with ENhance and exposed to XAR-2 Kodak film. The conversion of chloramphenicol to acetylated forms was quantified by two methods: autoradiographs were quantified by laser-assisted densitometry or by scraping the t.l.c. plates and measuring radioactivity by liquid scintillation spectroscopy. To correct for differences in transfection efficiencies, in most experiments the test constructs were co-transfected with a plasmid containing /,-galactosidase driven by the Rous sarcoma virus long-terminal repeat; fl-galactosidase activity was determined according to previously published procedures (Chang & Brenner, 1988). Additionally, to circumvent the intrinsic variation among different cell types, CAT assays were performed using constant amounts of total cellular protein, as determined by the Bradford assay (Bradford, 1976). RESULTS A collagen promoter/enhancer drives expression of a heterologous gene in a tissue-specific manner Plasmid pCOL-KT was generated by placing the 5'-noncoding and contiguous DNA spanning the first exon and intron of the proal(I) gene in front of the CAT gene, followed by removal of 410 bp (+ 68 to + 478) containing the first exon and the translation initiation site (Thompson et al., 1991). Conceivably, altered spatial relationships between the promoter and the first intron (due to a 410 bp deletion) in pCOL-KT, compared with the endogenous gene, could result in altered regulatory properties of such a construct. To circumvent the potential problem of artifactual expression of pCOL-KT, we had established earlier that the site of initiation of transcripts of endogenous proacl (I) and of pCOL-KT was identical (Thompson et al., 1991). Next, to test the transcriptional tissue specificity of the endogenous proal(I) gene promoter/enhancer, we transfected a variety of mesenchymal and non-mesenchymal cells and determined CAT activity by transient expression assays. A number of mesenchymal cell lines, including human fibroblasts (HFL- 1 and IMR-90) and a rhabdomyosarcoma cell line (A204), were transfected with pCOL-KT; parallel cultures were also transfected with pSV2CAT and pSVOCAT DNA to monitor expression of appropriate positive and negative controls. Expression of pCOL-KT occurred readily in HFL- I and A204 cells transfected with either pSV2CAT or pCOL-KT (Fig. 1); similar abundant expression of the reporter CAT gene was also seen in IMR-90 and NIH 3T3 cells and a number of human dermal and Vol. 286
181
synovial fibroblast lines (results not shown). Two cell lines of non-mesenchymal origin, a rat pheochromocytoma cell line (PC 12) and a human erythroleukaemia cell line (K562), which do not produce type I collagen, were also transfected with pCOLKT, pSVOCAT and pSV2CAT. While K562 and PC12 cells transfected with pSV2CAT, showed substantial exprepsion of CAT driven by the strong simian virus-40 promoter/enhancer, neither of these cells transfected with pCOL-KT expressed significant levels of CAT activity (Fig. 1). These data reveal that (i) the organization of the promoter-proximal and intronic regulatory sequences in pCOL-KT is compatible with the expression of appropriately initiated transcripts, and (ii) pCOL-KT expression occurs in a tissue-specific manner akin to the endogenous proal(I) gene. Although it seemed that sequence up to only 800 bp upstream of the transcription start site in pCOL-KT was sufficient for its expression in a tissue-specific manner, we were curious to investigate if other sequences further upstream were involved in the regulation of the proa I (I) gene. Thus we ligated an additional 1200 bp to pCOL-KT to create 'extended-promoter' pCOL-KT, [pCOL-KT(L)]. We found that although the patterns of tissuespecific expression of pCOL-KT(L) and pCOL-KT were identical, pCOL-KT(L) was expressed at significantly lower levels in a number of cell lines of mesenchymal origin, compared with pCOL-KT (H. Poppleton, J. Thompson & R. Raghow, unpublished work). Thus we elected to identify the cis-regulatory determinants of tissue specificity by site-specific alterations of pCOL-KT. A sequence located upstream of the proal(l) collagen gene promoter negatively regulates collagen gene expression The precise interactions among the transcriptional regulatory elements located in the 5'-upstream and intronic regions of the proal(I) gene remain somewhat unresolved and controversial. We reasoned that the two important sources of the discrepancies reported from different laboratories could be because either (i) (a)
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Fig. 1. The plasmid pCOL-KT is preferentially expressed in cells of mesenchymal lineage Two mesenchymal [HFL-1 (a) and A204 (b)] and two non-mesenchymal [K562 (c) and PC12 (d)] cell lines were transfected with pCOL-KT. Parallel cultures were not transfected with DNA (no DNA) or were transfected with a promoterless CAT plasmid (pSVOCAT) or with CAT driven by the simian virus 40 promoter/ enhancer (pSV2CAT). Cells were harvested 48 h after transfection and the cell extracts were assayed for conversion of '4C-labelled chloramphenicol into acetylated chloramphenicol by t.l.c. coupled with fluorography. pCOL-KT is readily expressed in HFL-1 and A204 cells and not expressed in K562 and PC12 cells. There is also abundant expression of pSV2CAT in all cell types regardless of their phenotype, while there is no expression of pSVOCAT in any of the cells.
C. P. Simkevich and others
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HFL-1 (a) or A204 (b) cells were transfected with restriction-endonuclease-derived deletion constructs Parallel cultures were also transfected with pSVOCAT Cell extracts were prepared and assayed for
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determine the fraction of "C-labelled chloramphenicol into its acetylated derivatives. Single deletions to -442 (EcoRV), -804 to -331 (XbaI) or
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the previously tested constructs had artifactually spatial organization of the promoter and intronic leading to an altered mode of expression compared endogenous gene, or (ii) the intrinsic variations test systems (e.g. Kenopus oocytes versus support transcription of the proal(I) gene yielded observations. It should be emphasized, however, potential problems are not mutually exclusive, bination of the two could yield the variations different laboratories. To circumvent the first problem, pCOL-KT, in which the relationship between 5'-upstream sequences and the first intron was similar endogenous proal(I) gene. In the following wished to test the functional consequence of series double deletions in the proal(() promoter/enhancer step in our analysis of the cis-regulatory elements representative analysis of the relative expression or double deletion constructs is shown in Fig. from several independent determinations are summarized 3. In both HFL- I and A204 cells, the EcoRV (lacking -625 to -442) and Xbal (nt -804 to -331) changed
sequences,
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of pCOL-KT showed significant decreases in CAT activity (2-10fold). Deletion of the SstII fragment in the intron (nt + 670 to + 1440) also led to a substantial decrease in expression compared with pCOL-KT. As anticipated, the XmaIII/SphI deletion mutant (nt -477 to -83) of pCOL-KT, which removed the canonical CAATT box located at -127, completely abolished expression of CAT (Fig. 2). Double deletion mutants of pCOL-KT were constructed by removing fragments of DNA after simultaneous digestion of the parent construct by two restriction enzymes (i.e. mixtures of EcoRV, XbaI and SstII), and the transcriptional activity of the deleted constructs was compared with that of pCOL-KT. Based on the representative data shown in Fig. 2, it appears that the negative impact of the single deletions in the promoter-proximal sequences was consistently relieved by deletion of the intronic sequences. For example, all three double deletion mutants (i.e. EcoV/SstII, XbaI/SstII and XbaI/SstII) were expressed at significantly greater levels compared with constructs containing only single deletions. Of all our constructs, the construct containing the XbaI*/SstII double deletion (lacking nucleotides -804 to -609 and + 670 to +1440) of pCOL-KT showed the most abundant expression: a 5-10-fold increase in CAT activity was observed in different experiments (results not shown). This remarkable effect of the XbaI*/SstII double deletions prompted us to test the effect of XbaI* deletion more extensively. We found that deletion with XbaI*, either alone or with the intronic SstII deletion, consistently boosted expression in all cell lines regardless of whether they were mesenchymal or non-mesenchymal in origin (see below).
that
experiments, of single
as
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of various
2, and
the
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Determinants of tissue specificity of the transcriptional control of the human proal(l) gene are distributed in the promoterproximal and intronic sequences The phenotypes of the various deletion constructs of pCOLKT strongly support the previously published observations (Rossouw et al., 1987; Bornstein et al., 1987, 1988; Bornstein & McKay, 1988; Liska et al., 1990) showing that sequences located upstream of the start of transcription and in the first intron determine optimal expression of human proal (I) collagen. Our observations extend earlier results by demonstrating the existence of an additional upstream negative regulatory element in the proal(I) promoter (e.g. 'XbaI* domain'; nucleotides -804 to -609). We tested whether the XbaI* domain was involved in determining the tissue-specific expression of the human proclI(I) gene. A number of different cell lines of non-mesenchymal origin were transfected with pCOL-KT and its various deletion derivatives, and CAT activity was determined after transient expression. The representative data for two of these cell lines, K562 and PC12, are shown in Fig. 4. While pCOL-KT was not detectably expressed in either cell line, deletion with XbaI* (nt -804 to -609) resulted in excellent expression of CAT activity in both K562 and PC12 cells. Although introduction of the SstII deletion in XbaI* constructs to create XbaI*/SstII (nt -804 to -609 and + 670 to +1440) curtailed expression (5-10-fold), expression of the latter was significantly greater than that of pCOL-KT (Figs. 3 and 4). Interestingly, the other two constructs containing double deletions (e.g. EcoRV/SstII and XbaI/SstII) also showed slight expression in K562 cells (Figs. 3 and 4) and in PC12 cells (results not shown). To determine the contribution of the intronic sequences to the apparent breakdown of tissue specificity of transcription as a result of the XbaI* deletion, we reintroduced the intronic SstII fragment in both positive and negative orientations in the construct containing the XbaI*/SstII deletion. The XbaI* construct, which contains the +670 to
+ 1440 SstII intronic fragment in its natural orientation, expressed CAT to much greater extent than did any of the single
1992
Transcriptional control of proal(I) collagen gene
183 pCOL-KT
HindilI
SstlI
Sstll
+1
-804
+1440
+670
+68 to +478
Hindill +1447
pCOL-KT
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,
CAT
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Xbal*
Relative CAT activity
HFL-1
A204
K562
PC-12
1.0
1.0
1.0
1.0
0.14±0.06 0.21±0.08
1.0
-
0.31 ± 0.04 0.21 ± 0.06
1.0
-
3.5 ± 0.21 2.9 ± 0.07
(-804 to -609)
Xmallil/Sphl
R t
