|Molecular Vision 2003;
Received 20 November 2002 | Accepted 31 December 2002 | Published 2 January 2003
The UV responsive elements in the human mimecan promoter: A functional characterization
Elena S. Tasheva,
Gary W. Conrad
Kansas State University, Division of Biology, Manhattan, KS
Correspondence to: Elena S. Tasheva, Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS, 66506-4901; Phone: (785) 532-6553; FAX: (785) 532-6653; email: firstname.lastname@example.org
Purpose: A major environmental stress encountered by humans is solar UV light, which can cause a spectrum of eye diseases, such as photokeratitis, cataract, pterygia, and ocular neoplasms. Mammalian defense mechanisms in response to adverse effects of UV light result in induction of a number of genes. Studies on the transcriptional regulation of genes that are expressed in the eye will increase understanding of both the physiological functions of these genes in the mammalian UV response, and the molecular bases for abnormalities associated with the above diseases. Mimecan is an extracellular matrix proteoglycan that is abundantly expressed in the cornea. The purpose of this study was to determine and characterize the UV responsive regulatory elements of the human mimecan promoter.
Methods: Transcriptional activity of the promoter was evaluated, before and after UV irradiation, using transient transfection of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Site directed mutagenesis and corresponding functional assays were used to determine the contribution of UV responsive regions to human mimecan transcription. Co-transfection experiments were used to investigate the role of transcription factors that bind these elements in the promoter and mediate the UV response. mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).
Results: The shortest promoter construct that was strongly activated following UV irradiation contained three initiator elements, an E-box element that is conserved between species, and the entire first intron of the human mimecan gene. Deletion of the intronic p53 binding site from this construct considerably diminished transcription and the UV response of the promoter. Surprisingly, deletion of the E-box sequence from this construct completely abolished both transcription and UV response of the promoter. These results demonstrated that the E-box is essential to transcription of the human mimecan gene and also is required for activation by p53. The role of the E-box, and the E-box binding protein, USF-1, in transcription and UV responses of the human mimecan promoter were confirmed by co-transfection experiments using dominant negative transcription factor, A-USF. In addition to these positive regulators, we demonstrate that the region between nucleotides -1314 and -1907 contains a transcriptional repressor site that is active in a time dependent manner following UV irradiation. Finally, we show that UV irradiation results in changes in mimecan mRNA levels in bovine corneal keratocytes in a time-dependent manner.
Conclusions: The human mimecan promoter contains several UV responsive regulatory elements that are conserved between human and bovine species and include the intronic p53 DNA binding site, the E-box in the proximal promoter, and the region between nucleotides -1314 and -1907. The E-box plays an important role in transcription and UV response of the human mimecan promoter. UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes and non-corneal cells.
Mimecan/osteoglycin is a corneal keratan sulfate proteoglycan (KSPG) that may play an essential role in the maintenance of corneal transparency and in the regulation of cellular growth in humans. Numerous observations over the past two decades support this notion. Thus, the absence, or an altered glycosylation state of corneal KSPGs have been reported in opaque corneal scars and in macular corneal dystrophy [1-4]. The genomic structure of mimecan is highly conserved between different species. Mimecan is a single copy gene, located on chromosome 9q22 in humans, that gives rise to multiple mRNA transcripts, three of which result from differential splicing within the first translated exon [5-7]. Yet, all mimecan mRNAs produce an identical protein that is conserved between mice, bovine and man, indicating its functional importance [8-10]. The expression of mimecan is tightly regulated in normal cells. The level of mimecan mRNA is high in corneal keratocytes maintained in low serum/serum free media, but rapidly decreases if these cells are grown in media containing serum . Growth factors and cytokines can modulate mimecan mRNA expression in corneal keratocytes and vascular smooth muscle cells [12,13]. The tumor suppressor protein p53 can activate transcription of the bovine mimecan gene . Furthermore, human mimecan mRNA is absent/low levels in a majority of tumors and cancer cell lines, where p53 frequently is inactivated/mutated [14,15].
As a transcription factor, p53 is activated in response to cellular stress, including the stress from UV induced cellular damage. In the damage response, p53 functions to activate genes that can either arrest cell cycle progression, providing sufficient time for repair of damaged DNA, or initiate apoptosis programmed cell death . To date, no direct connection between p53 and expression of the human mimecan gene in response to DNA damage has been established.
In addition to activation of p53, UV exposure also activates transcription factors AP-1 (activator protein 1), NF-κB (nuclear factor κB), USF-1 (upstream stimulatory factor 1), Oct-1 (octamer binding factor) and SRF (serum response factor) [17-21]. Interestingly, multiple potential DNA binding sites for all of the above transcription factors are present in the human mimecan promoter . As shown in Figure 1A, most of the DNA binding sites for these transcription factors are conserved in the bovine mimecan promoter and likely in other mammalian mimecan promoters. This is evidenced by the presence of the E-box in the mouse and rat mimecan 5'-flanking sequences. The E-box is a DNA binding site for several transcription factors known to play roles in cellular growth and stress responses. These include the evolutionarily conserved basic-helix-loop-helix-leucine zipper transcription factors USF-1, USF-2, and their differentially spliced isoforms, the Myc/Max/Mad family of transcription factors and Mitf (microphthalmia associated transcription factor) [22-24]. Recently, we demonstrated that USF-1, but not c-myc, binds this box in the human mimecan promoter .
In this study, we investigated the UV responsiveness of the human mimecan promoter and demonstrated functional roles for the conserved E-box in the proximal promoter, the intronic p53 DNA binding site, and the region between nucleotides -1314 and -1907. We also present evidence that USF-1 and p53 are transcriptional activators of the human mimecan promoter and that the E-box is required for activation by p53.
Plasmids and generation of the human mimecan deletion constructs
Expression plasmid pCX-USF1, containing USF-1 cDNA driven by the promoter of human cytomegalovirus (CMV), was kindly provided by Dr. Robert Roeder, Laboratory of Biochemistry and Molecular Biology, the Rockefeller University, New York, NY [22,25]. Expression plasmid A-USF, containing the USF dominant negative mutant (A-USF) coding sequence driven by the promoter of human CMV, was kindly provided by Dr. Charles Vinson, Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD [26,27].
The expression plasmid that contains wild type p53 cDNA driven by the promoter of human CMV and the reporter plasmid that contains a 3.5 kb fragment of the human mimecan promoter, including the first intron and 2.5 kb of the 5' flanking region, have been described previously [14,15]. This reporter plasmid was used as template for PCR amplifications to generate a series of deletion constructs of the human mimecan promoter. The primers and constructs, used in this study, are illustrated in Figure 1B. For convenience, numbering relative to the translation initiation start site was used (+1 in Figure 1B). The following primers were synthesized by Integrated DNA Technologies Inc. (Coralville, IA) and used in this study: Sense; Hm-2316; 5'-tctggtaccggacatatggtatctatccagc-3', Hm-1907; 5'-caaggtaccttgtatggtatctacaatgcc-3', Hm-1314; 5'-cacggtaccctagtacaacacactgcatttcaccc-3': Antisense; Hm-153; 5'-cgagggtgtgcgcagtaagg-3' and Hm-1146; 5'-atgtctgtgcattagccccaagtggg-3'. The sense primers contained a Kpn I site (marked in red) at their 5' end to facilitate positional cloning. The resultant DNA fragments were ligated into the Kpn I-Sma I site of the pGL3-basic firefly luciferase expression vector (Promega Corp., Madison, WI). The Quick Change Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA) was used to generate the human mimecan promoter constructs with deleted E-box and deleted p53 DNA binding sequences. The p-1314/-1146, p-2316/-153 and p-1314/-153 plasmids were used as templates to which the two primers containing the deleted E-box (HmdE sense; 5'-ctggcaaagatctctacgaaactgttc-3' and HmdE antisense; 5'-gaacagtttcgtagagaatctttgccag-3') or the two primers containing deleted p53 DNA binding sequence (Hmdp53 sense; 5'-gttttaaaactacttgtttctgttcatacc-3' and Hmdp53 antisense; 5'-ggtatgaacagaaacaagtagttttaaaac-3') were annealed and extended by Pfu I DNA polymerase according to the manufacturer's protocol. The extension products were treated with 10 U of Dpn I endonuclease at 37 °C for 1 h to digest the parental DNA template. The nicked vector DNAs incorporating the deleted E-box or deleted p53 DNA binding sequences were then transformed into E. coli XLI-Blue Supercompetent cells. Individual clones were isolated, purified using the Qiagen Plasmid kit (Qiagen Inc., Valencia, CA), and sequenced to confirm the deletion.
Cell culture, transient transfections, luciferase and β-galactosidase assays
Primary bovine keratocytes were isolated as described [5,10], plated in 6 well cluster plates (Corning Incorporated, Corning, NY), and incubated in Dulbecco's modified Eagle's medium/ nutrient mixture F-12 HAM (Sigma-Aldrich, St. Louis, MO), supplemented with 0.5% fetal bovine serum (FBS, Atlas Biologicals, Fort Collins, CO) and antibiotics (100 μg/ml each, penicillin and streptomycin). MG-63 cells were obtained from the American Type Culture Collection (Manassas, VA), and U2-OS and Saos-2 cells were a gift from Dr. H. Fattaey, Division of Biology, Kansas State University, Manhattan, KS. These cells were maintained in DMEM/F12, supplemented with 10% FBS and antibiotics. At 70% confluence, cells were transiently transfected using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol (12 μl reagent per 4 μg DNA). For each well, 2.5 μg of reporter gene and 250 ng of pSVβ-Gal plasmid (Clontech, Palo Alto, CA), to correct for transfection efficiency, were added. For co-transfection experiments, the indicated amounts of p53, pCX-USF1, or A-USF expression plasmids were used. An empty vector pBluescript (KS+/-, Stratagene, La Jolla, CA) was included to keep a total of 4 μg of DNA. Transfections with the pGL3 basic plasmid were used for background determination and transfections with the pGL3 control plasmid were used as positive controls. Identical transfections were performed in duplicate and repeated at least three times. Luciferase and β-galactosidase assays were performed as previously described . The results are reported as the means and standard errors from three separate transfections performed in duplicate.
After transfections (24 h), medium was removed and cells were exposed to 254 nm UV light (UVC) generated by a germicidal UV lamp at a dose of 20 J/m2. Fresh medium was added after UV exposure and cells were harvested for analysis at indicated intervals. Control cells were treated similarly but without UV exposure.
RNA isolation, reverse transcription-polymerase chain reaction
Total RNA was isolated from the cells at indicated intervals before and after UV irradiation using ToTALLY RNA Total RNA Isolation Kit (Ambion, Inc., Austin, TX). RNA (1 μg) was reverse transcribed using 10 μmol of the anchor primer oligonucleotide (dT)18, 0.5 mM dNTPs and Superscript II reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) for 50 min at 42 °C in buffer supplied by the manufacturer. The single stranded cDNA products (2 μl) were used as templates in a 50 μl PCR amplification reaction that contained 10 mM TRIS, pH 9, 50 mM KCl, 0.1% Triton X100 (v/v), 1.5 mM Mg2Cl, Taq polymerase (5 U), 0.2 mM dNTPs and 100 ng of each GSP, and carried out for 22-25 cycles (95 °C/45 s, 60 °C/1 min, 72 °C/2 min), to ensure linearity of amplification. Resulting PCR products were analyzed by electrophoresis on 2% agarose gels, with DNA visualized by ethidium bromide staining. The human mimecan cDNA (AF112465) was amplified using primers: Hm+2, 5'-tctcattcaccctcccacttgg-3' and Hm-1265, 5'-taatgcgtgagtcctgctgggttgg-3'. For amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), human control amplifier set from Clontech (Palo Alto,CA) was used (product number 5406-1). The bovine mimecan cDNA (M37974) was amplified using primers Bm+411; 5'-tttaggatccattaaaatgaagactctgcaatctacacttctcctg-3' and Bm-1313; 5'-gatagctcgagaatgtatgaccctatagg-3', designed to span the entire coding region of the gene and to contain BamH I and Xho I sites at their ends to facilitate the cloning and sequencing of amplified PCR fragment. The bovine lumican cDNA (L11063) was amplified using primers Bl+671; 5'-tgacttgagcttcaatcagatgacc-3' and Bl-1174; 5'-ccataaactgctgttccaggctacacc-3'. The bovine GAPDH cDNA (U85042) was amplified using primers Bgapdh+451; 5'-gtcatccatgaccactttggcatcgtgg-3' and Bgapdh-800; 5'-ttgaagtcgcaggagacaacctgg-3'.
Evidence for p53 dependent induction of human mimecan transcription
Recently, we demonstrated that p53 binds to its corresponding site in the first intron in vitro and trans-activates transcription of the bovine mimecan promoter in co-transfection experiments . To address the potential role of endogenous p53 induction in human mimecan gene expression, the following experiments were performed. U2-OS and Saos-2, human osteosarcoma cell lines, were treated with UV irradiation, or transfected with p53 expression vector and then treated with UV irradiation. Total RNA was extracted from control, p-53 transfected, and/or UV irradiated cells and the levels of mimecan and GAPDH (as an internal control) mRNAs were determined by RT-PCR. U2-OS cells were chosen because they do not express mimecan mRNA, although they are p53 positive [8,14,29]. The apparent "discrepancy" between the presence of p53 and the lack of mimecan expression in these cells can be explained by mdm2 gene amplification (murine double minute-2) and its increased translation in these cells [29,30]. MDM2 encodes a nuclear phosphoprotein that has been shown to interact with p53, thereby inhibiting p53 mediated transactivation in a dose dependent manner . UV irradiation is known to cause down regulation and rapid degradation of mdm2, as well as stabilization and increased half life of p53 protein . Therefore, if p53 is a transcriptional activator of the human mimecan gene, UV irradiation of U2-OS cells may result in increased and detectable levels of mimecan mRNA. Saos-2 cells were chosen because they are p53 negative, and they do not express mimecan mRNAs [8,15,32]. However, if p53 is introduced into these cells, and if this protein can activate transcription of the mimecan promoter, the corresponding mimecan mRNA should be detected in transfected cells. The results of these experiments are shown in Figure 2. UV irradiation of intact U2-OS and Saos-2 cells led to detection of mimecan mRNA in U2-OS (Figure 2B, U2-OS + UV), but not in Saos-2 (Figure 2C, Saos-2 + UV), results that demonstrate a correlation between the presence of p53 and mimecan expression. Transfections of U2-OS and Saos-2 cells with p53 expression vector, followed by UV irradiation (to increase the half life and protein stability of p53), led to further increase in the levels of mimecan mRNA in U2-OS cell (Figure 2B, U2-OS + p53 + UV), as well as to detection of mimecan mRNA in Saos-2 cells (Figure 2C, Saos-2 + p53 + UV). Together, these results demonstrate a positive correlation between the presence of p53 and mimecan expression and suggest a role for p53 as a transcriptional activator of the human mimecan gene.
UV responsive regulatory elements in the human mimecan promoter and significance of the E-box element to mimecan transcription
Next, we analyzed the UV responsiveness of the human mimecan promoter. A series of mimecan promoter/luciferase reporter constructs containing different lengths of the 5' flanking region of the gene were generated (Figure 1B). These constructs were transfected into primary bovine corneal keratocytes and luciferase expression was monitored before and after UV irradiation. Two constructs, p-2316/-1146 and p-1314/-1146, in which the 3' ends of the insert extended to the 5' untranslated region of the first exon, showed similar response in the presence and absence of UV irradiation (Figure 3). The slight decrease in luciferase activity 6 h after UV irradiation was followed by restored or slightly increased luciferase activities 24 h after UV iradiation. In contrast, the p-1907/-1146 construct, in which the region between nucleotides -2316/-1907 was deleted, showed a two fold decrease in luciferase activity compared to the p-2316/-1146 construct, and decreased luciferase activity was observed both without and after UV irradiation. Because all three constructs have identical 3' ends, together, these results demonstrate that (1) the region between nucleotides -2316 and -1907 contains a positive regulator of transcription and UV response in the mimecan promoter (Figure 3, compare activities of p-2316/-1146 to p-1314/-1146); (2) the region between nucleotides -1907 and -1314 contains a negative transcriptional regulator(s) that also is(are) UV responsive in a time dependent manner (Figure 3, compare luciferase activities of p-1907/-1146 before, 6 h and 24 h after UV irradiation to those of p-1314/-1146 and p-2316/-1146 constructs); and (3) The region between nucleotides -1314 and -1146 appears to be the minimal promoter region that can support transcription, but not the UV response, in the mimecan promoter.
The three constructs, p-2316/-153, p-1907/-153 and p-1314/-153, in which the 3' ends of the insert extended to the 5' untranslated region of the second exon, showed higher luciferase activities than the initial three constructs containing only the upstream promoter region. Consistent with our initial report, these results demonstrated the presence of an intronic enhancer . Following UV irradiation, activation of all three constructs was detected 24 h after UV (Figure 3, compare activities of p-2316/-1146 to p-2316/-153, p-1907/-1146 to p-1907/-153, and p-1314/-1146 to p-1314/-153 at 24 h after UV irradiation). The highest increase, 2.5 fold at 24 h after UV exposure, was observed with the shortest intron containing construct p-1314/-153. Because this construct contained both the intronic p53 DNA binding site and the conserved E-box element, these results indicate that the two sites serve as positive regulators to transcription and UV induction of the mimecan promoter. These results also are in agreement with the initial observations with p53 transfected and/or UV irradiated osteosarcoma cells. Interestingly, the p-1907/-153 construct, that also contained the entire first intron and the E-box motif, showed decreased luciferase activity at 6 h after UV irradiation. Since similar results were obtained with the p-1907/-1146 construct, in which the intronic sequences are missing but the upstream sequences are identical to those of the p-1907/-153 construct, these results again can be explained with the temporal negative regulatory effect of the upstream region (-1907/-1314).
To assess the significance of the E-box motif and the p53 DNA binding sites on transcriptional regulation of mimecan promoter, we generated constructs in which these motifs were deleted. These constructs were transfected into bovine corneal keratocytes and luciferase activities were determined before and after UV irradiation (Figure 4). Deletion of either the E-box or p53 site diminished transcription and the UV response of p-2316/-153 construct (in Figure 4, compare activities of p-2316/-153 to these of p-2316/-153dE and p-2316/-153dp53). Similarly, deletion of the E-box from a shorter construct, p1314/1146dE, diminished both transctiption and UV response of the promoter (in Figure 4, compare activities of p-1314/1146 to p-1314/-1146dE). Surprisingly, deletion of this E-box sequence from the intron containing p-1314/-153dE construct completely abolished both transcription and UV response of the promoter (Figure 4, compare activities of p-1314/-153 to those of p-1314/-153dE). Furthermore, co-transfections of p-1314/-153dE together with p53 expression vector resulted in no promoter activities with and without UV. Deletion of the p53 site in p-1314/-153dp53 construct considerably diminished but did not abolish transcription and UV response of the promoter (Figure 4, compare activities of p-1314/-153 to p-1314/-153dp53).
To extend these studies and to assess the significance of the E-box binding protein, USF-1, in human mimecan transcription, primary bovine keratocytes were co-transfected with human mimecan promoter constructs, together with expression vectors encoding the USF-1, or the dominant negative mutant of USF, A-USF. In the A-USF, the basic HLH domain of USF-1 is replaced with an acidic sequence, which stabilizes the heterodimers between A-USF and USF, resulting in inhibition of USF DNA binding [26,27]. The results from co-transfection experiments using bovine corneal keratocytes and A-USF, are shown in Figure 5A. In agreement with the initial results, co-transfections of p-2316/-153 or p-1314/-153 with A-USF led to decrease in luciferase activities with and without UV irradiation. To confirm these data in an independent system, we performed similar experiments using the human osteosarcoma cell line, MG-63. These cells, like Saos-2, fail to express wild type p53 . However, in contrast to Saos-2 and like bovine corneal keratocytes, MG-63 cells express mimecan mRNA constitutively and therefore are suitable for studies on transcriptional regulation of the mimecan promoter in a p53 deficient background . As shown in Figure 5B, co-transfections of MG-63 cells with p-2316/-153 or p-1314/-153 together with A-USF also led to decrease in luciferase activities of both constructs. In contrast, co-transfections of human mimecan promoter constructs, together with expression vectors encoding USF-1, led to two to three fold increases in luciferase activity in non-irradiated cells (Figure 6A and Figure 6B).
Taken together, the results described so far demonstrate that: (1) The E-box and the E-box binding protein, USF-1, are positive regulators of transcription. However, over expression of USF is not sufficient for an increase in the promoter response to UV; (2) In the absence of the E-box, transcription from the mimecan promoter can still occur (Figure 4, p-1314/-1146dE). The likely explanation may be that transcription initiates at alternative site(s), such as the Inr element. In addition to the E-box, there are three Inr elements within the region between -1314 and -1146 nucleotides (see Figure 1A); (3) The intronic p53 DNA binding site is also a positive regulator of transcription and UV response of the human mimecan promoter; (4) The E-box is required for activation by p53; and (5) The -1907/-1314 region mediated negative UV response appears to overcome the effects of positive regulators in a time dependent manner following UV irradiation.
UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes
Because the UV responsive regulatory elements are highly conserved between human and bovine mimecan promoters, next we sought to determine the expression of bovine mimecan in response to UV irradiation. Primary bovine corneal keratocytes were used for these studies. As shown in the semi-quantitative RT-PCR analysis in Figure 7, UV irradiation resulted in an initial decrease, followed by a 2.5 fold increase in the levels of mimecan mRNA, whereas levels of the housekeeping gene GAPDH were not changed significantly. For comparison, we also determined the expression of bovine lumican. Lumican is a small leucine rich proteoglycan that, similarly to mimecan, is expressed at high levels in the cornea . We chose to test the UV response of bovine lumican in addition to mimecan because the first intron of human lumican contains a half p53 DNA binding site similar to those shown in the 5' flanking region of bovine mimecan and within the first exon of mouse mimecan (Figure 1A), although the functional significance of these p53 half sites is presenty unclear. Interestingly, lumican mRNA levels also showed a decrease after UV irradiation followed by restored levels 48 h after UV exposure. However, this decrease was most prominent 24 h after UV treatment, whereas the decrease in mimecan mRNA level was most prominent 6 h after UV exposure and an increase was observed 24 h after UV treatment. The decrease, followed by an increase, in levels of the bovine mimecan mRNA after UV irradiation (Figure 7) could be accounted for by similar changes in transcriptional rates, changes in mRNA stability, alterations in nuclear processing of the nascent RNA molecules, or by combination of these processes. The role of changes in transcriptional rates is corroborated by two lines of evidence: (1) the presence of conserved UV responsive regions in the human and bovine mimecan promoters, an observation that suggests similar functional importance, as demonstrated for the conserved regulatory elements in other promoters and (2) results obtained by transient transfection assay utilizing human mimecan promoter/luciferase reporter constructs (Figure 3). As shown in Figure 3, a decrease in luciferase levels at 6 h after UV exposure was obtained with all constructs except for the shortest promoter construct p-1341/-153. Thus, the data shown in Figure 3 and Figure 7 suggest that the changes in mimecan mRNA levels are likely a result (at least in part) from changes in transcriptional rates. However, additional experiments will be necessary to determine the contribution of the factors described above to UV induced changes in the levels of mimecan mRNA. Such experiments include determination of mimecan mRNAs stabilities (there are 8 known bovine mimecan mRNA transcripts) and direct measurement of transcriptional activities by other methods, such as nuclear run-on assay.
In this study, we investigated the UV responsiveness of the human mimecan promoter and identified the regulatory elements that play roles in this response. We first verified the positive correlation between the presence of wild type p53 and mimecan expression using two osteosarcoma cell lines, U2-OS, which is p53 positive, and Saos-2, which lacks functional p53 (Figure 2). We then used transient transfections of the human mimecan promoter/luciferase reporter genes to define the regions of the promoter that play roles in the UV response. Three such regions that are highly conserved in the mimecan promoter were defined, one negative UV responsive region between nucleotides -1314 and -1907, and two positive regulatory sites, the E-box at position -1247 and the p53 DNA binding site located in the first intron.
As shown in Figure 1A, multiple binding sites for UV responsive transcription factors are present within the negative regulatory region, between nucleotides -1341 and -1907. Most of these sites are overlapping in the human mimecan promoter and, as demonstrated on other promoters, several transcription factors can bind to each one of these sites. For example, at least three proteins are known to bind to AP1 sites, dimers of c-Jun/c-Fos as well as dimers of c-Jun/ATF-2 [33,34]. In addition, a third transcription factor might be involved in these interactions, as shown for c-Jun and p53 in the regulation of the DNA repair gene, hMSH2, in response to UV . Similarly, besides SRF, several other proteins have been demonstrated to bind directly to the CarG-box (also known as the SRE, serum response element). These include SRE-BP, MyoD, AP1 and YY1. Interestingly, YY1 and MyoD have been shown to function as repressors of CarG-box activities [36-38]. Given these observations on other promoters, it is obvious that additional detailed studies will be necessary to determine the individual roles of each one of these sites in the UV response of the mimecan promoter.
In this study, the detailed characterization of the two positive regulatory elements, the p53 binding site and the E-box, was done by transient transfections into bovine corneal keratocytes and MG-63 cells. Deletion of the p53 site in two constructs, p-2316/-153dp53 and p-1314/-153dp53, resulted in decreased promoter activities with or without UV irradiation (Figure 4). These results are in agreement with the positive correlation between the presence of wild type p53 and mimecan expression in two osteosarcoma cell lines used in this study, U2-OS and Saos-2. They also are in agreement with the positive effect of p53 on bovine mimecan transcription that was reported previously . The important positive regulatory role of the E-box was demonstrated by two types of experiments: (1) deletion of the E-box element, which led to a decrease in promoter activities in two constructs, p-2316/-153dE and p-1314/-1146dE (Figure 4) and (2) co-transfection of mimecan promoter constructs, together with the dominant negative transcription factor A-USF, also led to decreases in promoter activities (Figure 5) or with the USF-1, that led to an increase in promoter activities (Figure 6). Furthermore, we demonstrate that the E-box is absolutely required for activation of the mimecan promoter by an intronic enhancer, such as the p53 site in bovine corneal keratocytes (Figure 4, p-1314/-153dE and p-1314/-153dE + p53).
In this report, we demonstrate that levels of bovine mimecan mRNA also undergo changes after UV irradiation. These changes are in agreement with the conclusions that the human mimecan promoter contains both the positive and the UV responsive negative regions that can be activated in a time dependent manner after UV irradiation.
USF proteins, similarly to p53, are ubiquitiously expressed transcription factors, and USF binding sites have been identified in a variety of gene promoters, including p53 itself . Because both p53 and USF-1 are known to regulate cell growth negatively, the data presented here raise the possibility that mimecan plays a role in the regulation of the same cellular processes in which p53 and/or USF are involved, i.e. adaptive responses, such as growth arrest and apoptosis. The initial observations on recently reported mimecan deficient mice demonstrate a role for mimecan in collagen fibrillogenesis . Functional challenges of these mice, as well as additional in vitro studies, will be necessary to determine the molecular mechanisms by which mimecan may participate in cellular adaptive responses. In this view it is of interest that lumican mRNA levels also undergo changes after UV irradiation. Because both mimecan and lumican are expressed at high levels in the cornea, where the core proteins undergo tissue specific glycosylation to become KSPGs in humans, it will be interesting to determine if this is associated with certain roles of corneal KSPGs in mammalian defense mechanisms in response to cellular stress (including UV light).
In conclusion, the present study demonstrates that the human mimecan promoter is UV responsive and establishes the critical role of transcription factors p53 and USF-1 as positive regulators of the human mimecan promoter. These results suggest a possible role of the corneal KSPG, mimecan, in the mammalian UV response.
The authors thank Dr. Roeder for providing the human USF-1 expression plasmid, Dr. Vinson for providing the expression plasmid A-USF, Dr. Abigail Conrad for critical reading of this manuscript, and members of Dr. Terry Johnson's laboratory for the regular supply of bovine corneas. This work was supported by NIH Grants EY13395 to GWC and EST and EY00952 to GWC.
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