Targeting myofibroblasts in model systems of fibrosis by an artificial alpha-smooth muscle-actin promoter hybrid.
ABSTRACT Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the -702 bp regulatory sequence of the alpha-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the beta-actin gene and the beta-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-beta-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-beta-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the -702 bp regulatory region of the SMA promoter linked to enhancing beta-actin and beta-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.
- SourceAvailable from: Jean-Jacques Meister[Show abstract] [Hide abstract]
ABSTRACT: The conjunctive presence of mechanical stress and active transforming growth factor beta1 (TGF-beta1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-beta1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-beta1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-beta1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-beta1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-beta1 and its receptor. We propose activation of TGF-beta1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.The Journal of Cell Biology 01/2008; 179(6):1311-23. · 9.69 Impact Factor
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ABSTRACT: Changes in the differentiated state of smooth muscle cells (SMCs) play a key role in vascular diseases, yet the mechanisms controlling SMC differentiation are still largely undefined. We addressed the role of basic helix-loop-helix (bHLH) proteins in SMC differentiation by first determining the role of two E-box (CAnnTG) motifs, binding sites for bHLH proteins, in the transcriptional regulation of the SMC differentiation marker gene, smooth muscle alpha-actin (SM alpha-actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a -2560- to 2784-bp SM alpha-actin promoter/LacZ reporter gene in vivo in transgenic mice. We then determined the potential role of class I bHLH proteins, E12, E47, HEB, and E2-2, in SM alpha-actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM alpha-actin promoter. Activation by HEB and E2-2 was synergistic with serum response factor. Additionally, the dominant-negative/inhibitory HLH proteins, Id2, Id3, and Twist, inhibited both the E12 and serum response factor-induced activations of the SM alpha-actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box-containing region of the SM alpha-actin promoter in vivo in the context of intact chromatin in SMCs. Taken together, these results provide the first evidence of E-box-dependent regulation of a SMC differentiation marker gene in vivo in transgenic mice. Moreover, they demonstrate a potential role for class I bHLH factors and their inhibitors, Id and Twist, in SM alpha-actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation.Circulation Research 06/2003; 92(8):840-7. · 11.09 Impact Factor
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ABSTRACT: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strain in vitro than in vivo. We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF) in vitro that were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loading in vivo and in vitro. We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1 h 45 min with the balance of time at rest to 6 h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1 Hz, 5% average elongation for 6 h, +/- PDGF-BB, IGF-I, TGF-beta 1, PTH, estrogen, PGE2, or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis. Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cells in vivo and in vitro will lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.Osteoarthritis and Cartilage 02/1999; 7(1):141-53. · 4.66 Impact Factor