A Smad3/FoxO3 Transcriptional Relationship in the Regulation of Vascular Growth

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Jake Francisco (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Cardiovascular disease (CVD) is a multi-faceted pathological condition that continually ranks as the number one cause of morbidity and mortality in the United States and worldwide. Unfortunately , despite extensive basic science and clinical investigation into mechanisms that can be targeted for therapeutic treatment of CVD , recent studies estimate the number of CVD-related deaths will continue to rise and is estimated that , by the year 2035 , more than 130 million American adults (>45%) will have some form of CVD. Additionally , the associated economic burden is expected to worsen as annual direct and indirect costs of CVD in the US alone are expected to surpass $1 trillion by 2035. Clearly , the healthcare and financial burdens of CVD continue to escalate and clinically treating these pathologies is of utmost importance. Uncontrolled , or synthetic , vascular smooth muscle cell (VSMC) proliferation and migration represent two primary events that are fundament for the development of CVD. Over the years a plethora of transcriptional , translational and post-translational factors with capacity to regulate vascular cell growth and phenotype has been identified. Of these agents , two transcription factors , Smad3 and FoxO3 , have become of recent interest due to their ability to markedly influence cell function (proliferation , migration , and apoptosis). Investigation into transforming growth factor (TGF)-[beta]1-directed Smad3 has shown proliferative effects of Smad3 in VSMCs , in turn promoting phenotypic switching to a synthetic , pro-growth character. Conversely , FoxO3 has been found to elicit a quiescent VSMC phenotype through induction of the cyclic-dependent kinase inhibitor (CdkI) p27. In addition to their implicated roles in the development or maintenance of CVD , it has been suggested that a dynamic relationship exists between Smad3 and FoxO3 transcription factors. Furthermore , considering the established relationship between Smad3 and FoxO3 and their disparate individual effects on VSMC proliferation and growth , further studies to elucidate this interaction in VSMC could reveal a therapeutic target for preventing or treating CVD. Our research broadly focuses on the relationship between Smad3 and FoxO3 in VSMCs , and our hypothesis is that FoxO3 exerts growth-suppressive actions via induction of cytostatic p21 and p27 expression and through mitigation of Smad3 signaling in VSMCs. Utilizing adenovirus expressing either GFP (as control; AdGFP) , Smad3 (AdSmad3) , or FoxO3 (AdFoxO3) , our findings in rat primary VSMCs demonstrate that overexpression of Smad3 induces a pro-growth phenotype with enhanced cell cycle progression , while overexpression of FoxO3 gives rise to growth-inhibition and cell cycle attenuation. Western blot analyses suggest that there are no significant differences in the expression of p21 or p27 compared to AdGFP controls. To explore this dynamic Smad3/FoxO3 relationship immunostaining for basal Smad3 and FoxO3 expression was performed on adherent VSMCs under quiescent or growth-stimulated conditions. Results show a significant increase in cytosolic FoxO3 , a decrease in cytosolic Smad3 , and an increase in nuclear Smad3 from quiescent to growth-conditions. To further characterize the cellular localization and activities of Smad3 and FoxO3 during growth , we performed cellular fractionation on adenovirus-infected VSMCs under quiescent or growth-stimulated conditions. In general , localization data on Smad3 and FoxO3 suggest that there is a dynamic relationship between these two transcription factors where environmental growth conditions dictate their location (and therefore , activity). Additionally , given the possibility that FoxO3 operates to traffic Smad3 to the cytoplasm as an inactivation/degradation step , we analyzed expression and activity of two FoxO3-directed ubiquitin ligases , Atrogin-1 and MuRF-1 , and the influence of Smad3. We found that AdSmad3/FoxO3 significantly reduced Atrogin-1 expression compared to GFP- , Smad3- , and FoxO3-treated cells , implying that FoxO3 operates to inhibit Smad3-mediated Atrogin-1 expression , and conversely that Smad3 acts to inhibit FoxO3-induced Atrogen-1 expression. Moreover , AdFoxO3 significantly increased MuRF-1 protein expression and promoter activity , both which were significantly reduced when coexpressed with Smad3. This investigation into Atrogin-1 and MuRF-1 as potential regulators of FoxO3's antagonistic effects on Smad3 provides support for these ubiquitin ligases , not only for the influence of FoxO3 on Smad3 but conversely for Smad3 influence on FoxO3. Collectively , these data provide evidence that a unique dynamic exists between the robust transcription factors Smad3 and FoxO3 in VSMCs. Data support the ability of FoxO3 to mitigate and normalize Smad3-driven cell proliferation and cell cycle progression. With Smad3 showing a propensity towards inducing a pathological and synthetic VSMC phenotype , FoxO3 could prove beneficial for the prevention and treatment of the root cause of these proliferative and occlusive vascular diseases.

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Thesis

Language: English

Date: 2019

Keywords

FoxO, FoxO3, Forkhead box O

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