A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA Considerable interest in the biology and pathophysiology of human malignant melanoma during the past two decades led to several efforts to delineate the structure and function of a melanoma-associated chondroitin sulfate proteoglycan (MCSP), uniformly expressed on >90% of human melanoma tissues and cultured cells (1-4). Biosynthetic and biochemical studies indicated that MCSP is a unique glycoproteinproteoglycan complex consisting of an N-linked glycoprotein of 250 kDa and a proteoglycan component >450 kDa. The 250-kDa molecule is the core glycoprotein of the proteoglycan, shown to be a chondroitin sulfate proteoglycan by its content of both 2-acetamido-2-deoxy-3-0-(f3-D-gluco-4-enepyranosyluronic acid)-f3-4-0-sulfo-D-galactose (ADi-4S) and 2-acetamido-2-deoxy-3-0-(13-D-gluco-4-enepyranosyluronic acid)-p3-6-0-sulfo-D-galactose (ADi-6S) chondroitin sulfate disaccharides (1, 2).Immunological and biochemical characterizations of MCSP demonstrated that the gene encoding it maps to human chromosome 15 (5) and that this major melanoma-associated antigen produced several murine mAbs because of its high expression and immunogenicity (6-8). Functional characterizations of MCSP indicated its role in growth control, adhesion, cell-substratum interactions, and cell-cell contacts (9,12). MCSP also proved to be an effective target for radioimaging of tumors of melanoma patients (13) and is currently used to target active specific immunotherapy with mouse anti-idiotypic mAb, which bear the internal image of antigenic determinants defined by anti-MCSP mAb (14). Conjugation of the anti-idiotypic mAb with a carrier and administration with an adjuvant induced humoral anti-MCSP immunity in about 60% of immunized patients with advanced melanoma (15), which was associated with statistically significant prolongation of survival (16). Because of the many interesting functional properties of MCSP, we report here the complete coding sequence and deduced amino acid sequence of its core protein. MATERIALS AND METHODSCell Lines and Antibody. The M21 (2) and A375 met (17) human melanoma cell lines and the Raji lymphoblastoid cell line (18) have been described previously, as has been the murine mAb 9.2.27 that specifically recognizes MCSP (1, 2). The UAC903 melanoma cell line was kindly provided by J. Trent (University of Michigan).Isolation of MCSP cDNA Clones. The protein sequence of the N-terminal end of MCSP (Y. Koyama and R.A.R., unpublished observations) revealed striking homology with the deduced amino acid sequence of the rat NG2 chondroitin sulfate proteoglycan (19). Thus, we used a cDNA fragment derived from the rat NG2 sequence (kindly provided by W. B. Stallcup, La Jolla Cancer Research Foundation, La Jolla, CA) as a prob...
We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.
The importance of insulin-like growth factor I (IGF-I) on maintenance of skeletal integrity has been widely recognized. Although osteoblasts secrete some IGF-I, the liver is the primary endocrine source for IGF-I. We have studied the regulation of the human IGF-I promoter in the hepatocyte cell line Hep3B, and we have shown that the IGF-I promoter, when co-transfected in Hep3B cells together with an estrogen receptor (ER)-␣ expression vector, was transcriptionally regulated by raloxifene or raloxifene-like molecules but not by 17-estradiol and 4(OH)-tamoxifen. The induction mediated by raloxifene is antagonized by 17-estradiol and mediated selectively by ER-␣, but not by ER-. Transfer of IGF-I promoter sequences from ؊733 to ؊65 or from ؊375 to ؊65 to a minimal Fos promoter resulted in a comparable responsiveness to raloxifene. This region contains two CAAT/enhancer-binding protein sites and an activator protein 1 site, both of which have been shown to be involved in estrogen receptor-mediated transactivation. When the CAAT/enhancer-binding protein sites were mutated in a construct bearing the sequence from ؊375 to ؊65 in front of the minimal Fos promoter, raloxifene induction was reduced, whereas mutation of the other elements did not affect induction. In addition, using chimeric proteins, we delineated the domains of ER-␣ that confer to ER-␣ transactivation abilities on the IGF-I promoter that are not exhibited by ER-. These data shed new light on the mechanism of action of antiestrogens and might help explain, at least in part, the bone-protective effects observed for some antiestrogens in ovariectomized animals.
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