Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
PGE2 and FGF-2 Upregulate Activities of the Human F-Spondin Promoter.
Attur1, Mukundan, Qing2, Yang, Wang3, Jinhua, Mix4, Kimberlee, Palmer1, Glyn, Abramson1, Steven B.
The extracellular matrix protein, F-spondin is a marker of both hypertrophic and osteoarthritic cartilage. Understanding the mechanisms that regulate its expression could therefore identify key pathways that regulate chondrocyte activity in development and disease. In this study we investigated transcriptional regulation of human F-spondin via the cloning and characterization of its 5' regulatory (promoter) region.
Genomic sequence containing the 5' regulatory region of F-spondin was obtained by PCR of human total genomic DNA (Clontech) using upstream primers designed from F-spondin genomic sequence available at the gene database www.ncbi.nlm.nih.gov, gene ID: 10418. PCR fragments were cloned into pGL3 Luciferase Reporter Vectors (Promega) for functional analysis. Luciferase assays were performed 36 h after transfection in the human chondrosarcoma cell line, SW1353.
An upstream 2.4 kb genomic sequence of human F-spondin was obtained by PCR, subcloned and sequenced bidirectionally. DNA sequence fidelity was confirmed using the NCBI blast alignment search tool. DNA sequence analysis revealed the presence of TATA box at position -74 and identification of transcription factor binding sites was performed using the JASPAR CORE database (http://jaspar.genereg.net/). High scoring putative binding sites within the 2.4 kb promoter region included NURR1 (NR4A2-nuclear receptor), NFAT, SOX-10 and CREB1. Functional promoter activity was assessed by transient transfection of pGL3 luciferase vectors encoding 2.4 kb (pFS-2.4Luc) and 0.5 kb (pFS-0.5Luc) of F-spondin upstream genomic sequence. In human chondrocytes, both pFS-2.4Luc and pFS-0.5Luc significantly increased luciferase activity above a pGL3 promoterless control vector, 20- and 70-fold, respectively. Since we have previously observed that F-spondin mRNA levels are induced by FGF-2 and PGE2 in OA chondrocytes, we examined their effects on promoter activity. PGE2 (10 uM) stimulated F-spondin luciferase activity 1020-fold for both 2.4 and 0.5 kb promoter constructs (p<0.001) above unstimulated controls. Cotransfection of a cDNA encoding NURR1, a transcription factor induced by PGE2 in human chondrocytes, also increased luciferase activity (100-fold) irrespective of promoter length. This is consistent with the presence of multiple putative NURR1 binding sites (~16) throughout the 2.5 kb promoter region. Conversely, FGF-2 (25 ng/ml) significantly increased luciferase activity of pFS-0.5Luc (2-fold; p<0.05) but not pFS-2.4Luc. This finding suggests that regulation of F-spondin via FGF-2 occurs via regulatory regions within its proximal 0.5 kb region.
Our results indicate that both developmental (FGF-2) and proinflammatory (PGE2) factors induce F-spondin expression in chondrocytes via discrete regions in its promoter. Sequence analysis and transfection studies suggest that NURR1 may mediate PGE2 induction of F-spondin via multiple binding sites in the promoter region. The current findings are consistent with previous observations demonstrating overlapping pro-inflammatory/catabolic effects of PGE2, NURR1 and F-spondin in OA chondrocytes.
To cite this abstract, please use the following information:
Attur, Mukundan, Qing, Yang, Wang, Jinhua, Mix, Kimberlee, Palmer, Glyn, Abramson, Steven B.; PGE2 and FGF-2 Upregulate Activities of the Human F-Spondin Promoter. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :61