Proteomic analysis of potential keratan sulfate, chondroitin sulfate A, and hyaluronic acid molecular interactions.

PURPOSE: Corneal stroma extracellular matrix (ECM) glycosaminoglycans (GAGs) include keratan sulfate (KS), chondroitin sulfate A (CSA), and hyaluronic acid (HA). Embryonic corneal keratocytes and sensory nerve fibers grow and differentiate according to chemical cues they receive from the ECM. This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs. METHODS: Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared and used in microarray protocols to assess their interactions with 8268 proteins and a custom microarray of 85 extracellular epitopes of nerve growth-related proteins. Surface plasmon resonance (SPR) was performed with bKS and SLIT2, and their ka, kd, and KD were determined. RESULTS: Highly sulfated KS interacted with 217 microarray proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 proteins, including several ECM-related structural proteins. Of 85 ECM nerve-related epitopes, KS bound 40 proteins, including SLIT, 2 ROBOs, 9 EPHs, 8 Ephrins (EFNs), 8 semaphorins (SEMAs), and 2 nerve growth factor receptors. CSA bound nine proteins, including ROBO2, 2 EPHs, 1 EFN, two SEMAs, and netrin 4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. The KS core protein mimecan/osteoglycin bound 15 proteins. CONCLUSIONS: Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.

Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass.

Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus1 and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of 22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosis.

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice.

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.

Expression and localization of leucine-rich B7 protein in human ocular tissues.

PURPOSE: Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells. METHODS: Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize. CONCLUSIONS: The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.

Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS.

PURPOSE: To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS). METHODS: Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4- and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes. Digest solutions were analyzed directly by ESI-MS/MS. RESULTS: The concentration of KS monosulfated disaccharide (MSD) Gal-beta-1,4-GlcNAc(6S) in E8 cornea equaled that at E20, declined to its lowest level by E10, increased to a second peak by E14, decreased to a second low by E18, peaked again by E20, and remained high in adult corneas. A similar concentration profile was observed for KS disulfated disaccharide (DSD) Gal(6S)-beta-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD was higher than that of MSD from E8 to E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS Deltadi-4S plus Deltadi-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of Deltadi-6S is highest at E8, then decreases through development as the concentration and molar percent of Deltadi-4S increases from E8 and exceeds that of Deltadi-6S after E14. CONCLUSIONS: New, rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development.

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry.

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.

Analysis of transcriptional regulation of the small leucine rich proteoglycans.

PURPOSE: Small leucine rich proteoglycans (SLRPs) constitute a family of secreted proteoglycans that are important for collagen fibrillogenesis, cellular growth, differentiation, and migration. Ten of the 13 known members of the SLRP gene family are arranged in tandem clusters on human chromosomes 1, 9, and 12. Their syntenic equivalents are on mouse chromosomes 1, 13, and 10, and rat chromosomes 13, 17, and 7. The purpose of this study was to determine whether there is evidence for control elements, which could regulate the expression of these clusters coordinately. METHODS: Promoters were identified using a comparative genomics approach and Genomatix software tools. For each gene a set of human, mouse, and rat orthologous promoters was extracted from genomic sequences. Transcription factor (TF) binding site analysis combined with a literature search was performed using MatInspector and Genomatix' BiblioSphere. Inspection for the presence of interspecies conserved scaffold/matrix attachment regions (S/MARs) was performed using ElDorado annotation lists. DNAseI hypersensitivity assay, chromatin immunoprecipitation (ChIP), and transient transfection experiments were used to validate the results from bioinformatics analysis. RESULTS: Transcription factor binding site analysis combined with a literature search revealed co-citations between several SLRPs and TFs Runx2 and IRF1, indicating that these TFs have potential roles in transcriptional regulation of the SLRP family members. We therefore inspected all of the SLRP promoter sets for matches to IRF factors and Runx factors. Positionally conserved binding sites for the Runt domain TFs were detected in the proximal promoters of chondroadherin (CHAD) and osteomodulin (OMD) genes. Two significant models (two or more transcription factor binding sites arranged in a defined order and orientation within a defined distance range) were derived from these initial promoter sets, the HOX-Runx (homeodomain-Runt domain), and the ETS-FKHD-STAT (erythroblast transformation specific-forkhead-signal transducers and activators of transcription) models. These models were used to scan the genomic sequences of all 13 SLRP genes. The HOX-Runx model was found within the proximal promoter, exon 1, or intron 1 sequences of 11 of the 13 SLRP genes. The ETS-FKHD-STAT model was found in only 5 of these genes. Transient transfections of MG-63 cells and bovine corneal keratocytes with Runx2 isoforms confirmed the relevance of these TFs to expression of several SLRP genes. Distribution of the HOX-Runx and ETS-FKHD-STAT models within 200 kb of genomic sequence on human chromosome 9 and 500 kb sequence on chromosome 12 also were analyzed. Two regions with 3 HOX-Runx matches within a 1,000 bp window were identified on human chromosome 9; one located between OMD and osteoglycin (OGN)/mimecan genes, and the second located upstream of the putative extracellular matrix protein 2 (ECM2) promoter. The intergenic region between OMD and mimecan was shown to coincide with different patterns of DNAse I hypersensitivity sites in MG-63 and U937 cells. ChiP analysis revealed that this region binds Runx2 in U937 cells (mimecan transcript note detectable), but binds Pitx3 in MG-63 cells (expressing high level of mimecan), thereby demonstrating its functional association with mimecan expression. Upon comparing the predictions of S/MARs on the relevant chromosomal context of human chromosomes 9 and 12 and their rodent equivalents, no convincing evidence was found that the tandemly arranged genes build a chromosomal loop. CONCLUSIONS: Twelve of 13 known SLRP genes have at least one HOX-Runx module match in their promoter, exon 1, intron 1, or intergenic region. Although these genes are located in different clusters on different chromosomes, the common HOX-Runx module could be the basis for co-regulated expression.

Analysis of the expression of chondroadherin in mouse ocular and non-ocular tissues.

PURPOSE: Chondroadherin (Chad) is a unique member of the small leucine rich repeat proteoglycan gene family. It is expressed at high levels in certain zones of cartilage and also has been detected in bone, tendon, bone marrow, and chondrosarcoma cells. Recently, we demonstrated that Chad is expressed in human and mouse lens and that its expression is decreased in the absence of mimecan/osteoglycin. This finding prompted us to explore the expression of Chad in other ocular and non-ocular tissues. METHODS: Reverse transcription-polymerase chain reaction amplification (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used to determine the expression of Chad in human and mouse tissues. RESULTS: RT-PCR analysis showed strong expression of Chad in mouse brain, heart, lung, and embryo. Moderate expression was detected in mouse thymus, spleen, testis, and ovary, and very low expression in kidney and liver. Chad was highly expressed in human cornea, brain, and skeletal muscle, and moderately in human retina and lens. By ISH, Chad mRNA was detected in cornea, lens, and retina of postnatal day 21 mouse eyes. By IHC, immunostaining for Chad was seen in epithelial and endothelial layers of the cornea, as well as in lens, ciliary body, and retina of the adult mouse eye. Strong immunostaining was noted in retinal rod, cone, and plexiform layers. IHC analysis of tissue microarray demonstrated presence of Chad in brain (cerebellum), skeletal and cardiac muscles, lung, gastrointestinal tract, ovary, and cartilage. In most tissues, Chad expression was associated with either peripheral nerves and/or blood vessels. In the stomach and intestines, positive immunostaining was observed in Meissner's plexus and enteroendocrine cells. Intriguingly, positive immunostaining also was observed in Purkinje cells of the cerebellum and pancreatic islets. CONCLUSIONS: The present work establishes the expression and localization of Chad in several new locations including cornea, lens, ciliary body, retina, cerebellum, pancreatic islet, blood vessels, and peripheral nerves. The surprisingly broad expression pattern of Chad suggests potential roles for this protein in cell specific and/or tissues specific function(s). The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of Chad in health and disease.

Differentially expressed genes in the lens of mimecan-null mice.

PURPOSE: Members of the small leucine-rich proteoglycans (SLRP) gene family are essential for normal collagen fibrillogenesis in various connective tissues and important regulators of cellular growth, differentiation, and tissue repair. Mimecan is a member of this gene family and is expressed in many connective tissues. We have previously reported that knockout of the mouse mimecan gene results in abnormal collagen fibrillogenesis, mainly in the cornea and skin. During the course of our studies on biological roles of mimecan in the eye, we found that this gene is expressed in the mouse lens. Here, we sought to identify gene expression changes in the lens that are associated with the absence of mimecan. METHODS: Reverse transcription-polymerase chain reaction amplification (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used to determine mimecan expression in human and mouse eyes. Microarray hybridization was used to determine gene expression differences between lenses isolated from mimecan-null and wild type mice. Relative quantitative RT-PCR was used to verify the expression levels of a subset of the identified genes. RESULTS: By ISH and IHC, mimecan mRNA was detected in cornea and lens at embryonic day 16.5 (E16.5) and postnatal day 10 (P10) mouse eyes. By RT-PCR, mimecan mRNA was detected in human cornea, lens, iris, and retina. In mimecan-null mice lenses, microarray analysis of 5,002 mouse genes demonstrated a more than two fold increase in expression of 65 genes and a more than two fold decrease in expression of 76 genes. Among genes with increased expression were cell adhesion molecules, G-protein coupled receptors, intracellular signaling molecules, genes involved in protein biosynthesis and degradation, and genes involved in immune function. Decreased expression was found in extracellular matrix molecules, calcium binding and transporting proteins, and genes known for their roles in regulating cellular motility. Intriguingly, decreased gene expression was observed with two SLRP family members, biglycan and condroadherin, as well as with several stress-response proteins, including gammaA-crystallin, hemoglobin alpha 1, and metallothionein 1. Quantitative RT-PCR confirmed changes in expression of 12 genes selected from the arrays. CONCLUSIONS: In this report we present the first demonstration that mimecan is constitutively expressed in the vertebrate lens. The results from gene expression profiling reveal the ability of mimecan to influence expression of biglycan and chondroadherin, thereby indicating possible novel regulatory interactions between these SLRP family members. As with mimecan, the expression of chondroadrein in vertebrate lens has not been reported previously. Our results provide insight into the function of mimecan in the lens and enable further characterization of molecular mechanisms by which this protein exerts its biological roles.

Interferon-gamma regulation of the human mimecan promoter.

PURPOSE: The human mimecan/osteoglycin promoter contains multiple interferon-stimulated response elements (ISRE) and interferon-gamma-activation sites (GAS). ISRE and GAS motifs are present in a variety of interferon (IFN)-inducible genes. The purpose of this study was to investigate whether IFN-gamma affects mimecan gene expression and, if so, to determine the cis-elements and transcription factors that mediate its action. METHODS: Electrophoretic mobility shift assay (EMSA) was used to investigate whether nuclear proteins from IFN-gamma-treated cells bind to regions of the human mimecan promoter containing ISRE sites. Incubation of nuclear extracts with specific antibodies was used to identify transcription factors that bind to these sites. Transcriptional activity of the promoter was evaluated by transient transfections of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Co-transfection experiments were used to study the role of transcription factors that bind ISRE elements in the promoter and mediate the IFN-gamma response. Expression of mRNAs was analyzed by reverse transcription-polymerase chain reaction. RESULTS: Using probes that correspond to two ISRE sites located in the first intron of the human mimecan gene, we detected specific DNA-protein complexes with nuclear extracts from IFN-gamma-treated cells. Formation of DNA-protein complexes was abrogated by competition with unlabeled probe and one of the complexes was supershifted by the anti-interferon regulatory factor-1 (IRF-1) antibody. Interestingly, when probe that corresponds to a conserved E-box (CACATG) in the proximal promoter and nuclear extracts from IFN-gamma-treated cells were used in EMSA, increased binding of upstream stimulatory factor-1 (USF-1) was observed. Co-transfection of a mimecan promoter construct that contained the entire first intron with IRF-1, or with both IRF-1 and USF-1 expression plasmids, suppressed luciferase activity of the promoter in corneal keratocytes and T-47D cells. In contrast, co-transfection experiments with IRF-2, or with both IRF-2 and USF-1, led to increased luciferase activity of the same promoter construct. RT-PCR analyses demonstrate that IFN-gamma rapidly and transiently suppresses mimecan expression and induces IRF-1 and IRF-2 mRNAs in bovine corneal keratocytes. CONCLUSIONS: IRF-1 binds to ISRE sites, located in the first intron of the human mimecan gene, and negatively regulates mimecan expression at the level of transcription. Consistent with these observations, an inverse correlation between the expression of mimecan and IRF-1 in bovine corneal keratocytes and non-corneal cells was demonstrated. IRF-2 positively regulates mimecan transcription in corneal keratocytes. Because the intact E-box in the proximal promoter was required for IRF-1 and IRF-2 effects on mimecan transcription, potential direct or indirect interactions between USF-1 and IRF-1 and IRF-2 are likely.

The UV responsive elements in the human mimecan promoter: a functional characterization.

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-deficient mice have collagen fibril abnormalities.

PURPOSE: To study the role of mimecan, a member of the small leucine-rich proteoglycans (SLRPs) gene family and one of the major components of the cornea and other connective tissues, mice that lack a functional mimecan gene were generated and characterized. METHODS: Mimecan-deficient mice were generated by gene-targeting using standard techniques. Mice were genotyped by Southern blot analysis. The absence of mimecan transcripts was confirmed by Northern blot analysis. Corneal clarity was examined by slit lamp biomicroscopy. The strength of the skin was evaluated using a biomechanical skin fragility test. Collagen morphology in cornea and skin preparations from mimecan-null and control wild-type mice was analyzed by transmission electron microscopy. The diameter of collagen fibrils in these tissues was determined by morphometric analysis. RESULTS: Mice lacking mimecan appear to develop normally, are viable and fertile. In a controlled laboratory environment they do not display an evident pathological phenotype compared to wild type mice. Examination of corneal clarity and measurements of corneal thickness show no significant changes in the cornea. However, a skin fragility test revealed a moderate reduction in the tensile strength of skin from mutant mice. Ultrastructural analyses show, on average, thicker collagen fibrils in both corneal and skin preparations from mimecan-null mice. Collagen fibrils from the cornea of mutant mice show an average diameter of 31.84+/-0.322 nm, versus 22.40+/-0.296 nm in their wild type litter-mates. The most pronounced increase in collagen fibril diameter was found in the skin of mimecan-null mice, who demonstrated an average diameter of 130.33+/-1.769 nm, versus 78.82+/-1.157 nm in the wild type mice. In addition, size variability and altered collagen morphology was detected in dorsal and tail skin preparations from the mutant mice. CONCLUSIONS: The results of the present study demonstrate that mimecan, similar to other members of the SLRP gene family, has a role in regulating collagen fibrillogenesis in vivo. Further studies, such as functional challenges, an evaluation of potential compensation by other proteins (including members of the SLRP family), and generation of double-knockouts will be necessary to fully uncover physiological functions of mimecan in mice.

Analysis of the promoter region of human mimecan gene.

Mimecan is a small leucine-rich proteoglycan (SLRP) that may play an important role in the regulation of cellular growth as illustrated by ability of growth factors and cytokines to modulate its expression and by recent demonstration that bovine mimecan is transcriptionally activated by p53 through a conserved intronic recognition site. To investigate transcriptional regulation of human mimecan, the upstream region and the first intron of this gene were cloned and analyzed. Within a 296-bp upstream region required for basal gene expression, there are three initiator (Inr) elements, an E-box and Oct-1, metal response element (MRE), and NF-kappa B recognition sites. Upstream stimulatory factor (USF)-1, Oct-1 and MRE-binding proteins were identified as proteins that bind to these regulatory elements and support transcription of mimecan in MG-63 cells. The first intron of human mimecan contains enhancer and silencer elements. Reporter gene transfections demonstrated that cooperation of upstream region and intronic enhancer elements are required for maximal gene expression in both p53-deficient and wild-type p53-expressing cells. Within the footprinted intronic enhancer region an interferon-stimulated response element (ISRE) is present. Using electrophoretic mobility shift assay (EMSA), interferon regulatory factor (IRF)-1 was identified as a protein that binds to this region in MG-63 but not in U-937 cells. In vitro translated IRF-1 also was shown to bind to this ISRE. These results demonstrate that the first intron of human mimecan gene carries important regulatory elements, including p53 DNA-binding site and ISRE, and should promote a better understanding of molecular bases for cell type-specific regulation of mimecan transcription.