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Dynamic interplay between activators and repressors of smooth muscle alpha-actin gene transcription during myofibroblast differentiation

Hariharan, Seethalakshmi

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Myofibroblasts are the primary cellular mediators of wound-healing that provide contractile force required for wound contraction, closure, and restoration of tissue integrity. De novo expression of smooth muscle alpha-actin (SMA) is a physiologically relevant process required for assembly of thin filaments and generation of contractile force by myofibroblasts. Chronic myofibroblast differentiation and associated excessive secretion of type I collagen paves the way to the emergence of fibrocontractive disorders that eventually lead to cardiopulmonary remodeling and dysfunction. Transforming growth factor beta-1 (TGF beta-1) is the major profibrotic agonist that drives myofibroblast differentiation in healing wounds. Characterization of transcriptional control of the SMA gene has led to the identification of cis-regulatory DNA elements within the 200 bp SMA proximal core promoter as well as corresponding trans-acting proteins that modulate SMA gene output. Dynamic interplay between transcriptional activators and repressors is implicated as the major means for governing SMA gene transcription during TGF beta-1-mediated myofibroblast differentiation. Based on previous biochemical and molecular biological studies, we hypothesized that the purine-rich element (Pur) binding proteins alpha and beta (Pur alpha and Pur beta) may negatively modulate the DNA-binding activities of serum response factor (SRF) and TGF beta-1-regulated Smad3 transcriptional activators with SMA promoter DNA during the process of myofibroblast differentiation. Comparison of SMA protein expression in human pulmonary fibroblasts (hPFB) cultured on a rigid plastic substrate or softer substrates prepared from native or denatured forms of type I collagen indicated that SMA expression was highly suppressed on collagenous substrates and was associated with an enhanced ability of Pur alpha/beta repressor proteins to interact with the SMA promoter. These results indicated that Pur alpha/beta may play a significant role in the negative regulation of SMA expression during myofibroblast differentiation. Comparative DNA-binding analysis indicated that Pur beta had a stronger affinity for the SMA core promoter and was highly effective in displacing equimolar quantities of Pur alpha from the DNA. Nuclear extracts prepared from quiescent fibroblasts and TGF beta-1-stimulated myofibroblasts (MFBs) cultured on rigid plastic were titrated against varying quantities of recombinant Pur alpha or Pur beta to discern their effect on SRF and pSmad3 interaction with the core promoter. Pur alpha was capable of potentiating and stabilizing SRF interaction with the SMA promoter in quiescent fibroblasts, while prior association of Pur alpha with promoter DNA negatively regulated the formation of SRF:DNA complexes in both quiescent and TGF beta-1-stimulated fibroblasts. In contrast, Pur beta generally antagonized SRF action in quiescent fibroblasts although with slightly reduced potency in TGF beta-1-stimulated MFBs. The ability of pSmad3 to interact with the SMA promoter was unaffected by Pur alpha when pSmad3 had access to DNA first, but not when excess Pur alpha was pre-bound to DNA. On the other hand, Pur beta was incapable of affecting pSmad3-DNA interaction during the early time periods after TGF beta-1 stimulation, while higher quantities of Pur beta disrupted pSmad3 interaction with DNA later on indicating TGF beta-1 treatment time-dependent modulation of Pur protein-pSmad3-DNA interplay. Since gene expression is also dependent on changes in protein interaction profiles of transcription factors, we examined formation of protein complexes containing SRF/Smad3 activators, the SRF co-activator called MRTF-A, and Pur alpha/beta repressors. SRF-Pur alpha/beta protein complexes present in quiescent fibroblasts dissociated in response to a TGF beta-1 stimulus. On the other hand, pSmad3 and nuclear MRTF-A were coupled with Pur beta during the early phase of TGF beta-1 signaling, but associated with Pur alpha during later TGF beta-1 time points. Finally, we showed that combinations of Pur alpha/beta and YB-1 repressor proteins co-operatively downregulated SRF and pSmad3 DNA-binding activities in quiescent fibroblasts and TGF beta-1-activated myofibroblasts, respectively. In support of these biochemical studies, immunohistochemical analysis of Pur beta and SMA in transgenic mouse lung tissue engineered to overexpress active TGF beta-1 showed a correlation between a TGF beta-dependent dispersion of nuclear Pur beta and increased expression of SMA in interstitial fibroblasts pointing to a possible inverse correlation between nuclear Pur beta availability and fibrosis progression. In summary, we show for the first time that the DNA-binding activities of SRF and pSmad3 can be differentially modulated by Pur alpha and Pur beta while the concomitant presence of Pur alpha/beta and YB-1 consistently attenuated SRF- and pSmad3-DNA interaction efficiently indicating possible co-operativity among the transcriptional repressors in control of myofibroblast gene expression. Understanding nucleoprotein interactions may prove useful in the design of therapeutic approaches to effectively target dysregulated myofibroblast differentiation and avoid progression to organ-destructive fibrotic disease.
Arthur Strauch, PhD (Advisor)
Daren Knoell, PhD (Committee Member)
Beth Lee, PhD (Committee Member)
Susheela Tridandapani, PhD (Committee Member)
184 p.

Recommended Citations

Citations

  • Hariharan, S. (2014). Dynamic interplay between activators and repressors of smooth muscle alpha-actin gene transcription during myofibroblast differentiation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396440406

    APA Style (7th edition)

  • Hariharan, Seethalakshmi. Dynamic interplay between activators and repressors of smooth muscle alpha-actin gene transcription during myofibroblast differentiation . 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1396440406.

    MLA Style (8th edition)

  • Hariharan, Seethalakshmi. "Dynamic interplay between activators and repressors of smooth muscle alpha-actin gene transcription during myofibroblast differentiation ." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396440406

    Chicago Manual of Style (17th edition)