Abstract
Abnormal transforming growth factor-beta (TGF-beta) signaling is a critical contributor to the pathogenesis of various human diseases ranging from tissue fibrosis to tumor formation. Excessive TGF-beta signaling stimulates fibrotic responses. Recent research has focused in the main on the antiproliferative effects of TGF-beta in fibroblasts, and it is presently understood that TGF-beta-stimulated cyclooxygenase-2 (COX-2) induction in fibroblasts is essential for antifibroproliferative effects of TGF-beta. Both TGF-beta and COX-2 have been implicated in tumor growth, invasion, and metastasis, and therefore tumor-associated fibroblasts are a recent topic of interest. Here we report the identification of positive and negative regulatory factors of COX-2 expression induced by TGF-beta as determined using proteomic approaches. We show that TGF-beta coordinately up-regulates three factors, heterogeneous nuclear ribonucleoprotein A/B (HNRPAB), nucleotide diphosphate kinase A (NDPK A), and nucleotide diphosphate kinase A (NDPK B). Functional pathway analysis showed that HNRPAB augments mRNA and protein levels of COX-2 and subsequent prostaglandin E(2) (PGE(2)) production by suppressing degradation of COX-2 mRNA. In contrast, NDPK A and NDPK B attenuated mRNA and protein levels of COX-2 by affecting TGF-beta-Smad2/3/4 signaling at the receptor level. Collectively, we report on a new regulatory pathway of TGF-beta in controlling expression of COX-2 in fibroblasts, which advances our understanding of pathophysiological mechanisms of TGF-beta.
Highlights
Cellular matrix production [1,2,3,4,5]
Among the wide range of biological functions regulated by TGF-, its effect on fibrosis remains one of the most extensively studied research areas because excessive TGF- signaling is a critical contributor to the pathogenesis of various fibrotic diseases [2, 7]
nucleotide diphosphate kinase A (NDPK A) and NDPK B Impair TGF--dependent Induction of COX-2 Not at the Transcriptional Level but at the TGF- Receptor Level—With these apparently conflicting results, we conducted reporter assays similar to that used in a previous report to rule out cell typedependent effects, which showed that NDPK A and NDPK B can transactivate the Cox-2 promoter
Summary
Cell Culture—Murine 10T1/2 fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10%. After 24 h of growth arrest with serum-free medium, subconfluent 10T1/2 cells were stimulated with recombinant human TGF-1. A differential display protein expression map for TGF--treated and -untreated cells. After 24 h of growth arrest with serum-free medium, subconfluent cells were stimulated with recombinant human TGF-1 (1 ng/ml) for 12 h and harvested. The center panel is a two-dimensional differential map of the protein expression of the treated versus untreated cells. Consider- were automatically and sequentially applied to a high perforing that up-regulation of COX-2 and the subsequent prosta- mance reversed phase column as the second dimension separaglandin cascade play crucial roles in tumorigenesis, tumor inva- tion, which consists of a linear gradient of 0 –100% B against A sion, and metastasis [15], TGF--induced COX-2 expression in for 30 min, where A is 0.1% trifluoroacetic acid in water and B is fibroblasts is envisioned to be a critical contributory factor in 0.08% trifluoroacetic acid in acetonitrile. Our findings advance our understanding of the underlying Tips (Millipore) and eluted with 70% acetonitrile and 0.1%
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