Abstract
The histone deacetylase (HDAC) inhibitors, butyrate and trichostatin A (TSA), are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC) that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi) by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.
Highlights
Atherosclerosis and restenosis, complex pathologies of medium to large blood vessels, are multifactorial vascular proliferative diseases responsible for the global burden of cardiovascular diseases
While the effects of butyrate and trichostatin A (TSA) on cdkI, p21Cip1, one of the genes that are universally upregulated in HDAC inhibitors (HDACi)-induced proliferation arrest [11,16,17,33], and on cyclin-D-specific cdk4, cdk6, cyclin E-specific cdk2, p15INK4b and phosphorylation/activity state of Rb protein fits the portfolio for arrest of proliferation, their similar effects on cyclin D3 and differential effects on cyclin D1 is intriguing
To assess whether changes in stability/degradation contributes to the differences in the cyclin D1 levels in vascular smooth muscle cells (VSMC) treated with butyrate and TSA, phosphorylated cyclin D1 levels are determined by western analysis with an antibody specific to phosphorylated Threonine residue at position 286 (Thr-286) cyclin D1
Summary
Atherosclerosis and restenosis, complex pathologies of medium to large blood vessels, are multifactorial vascular proliferative diseases responsible for the global burden of cardiovascular diseases. VSMC proliferation, the hallmark of vascular proliferative diseases, involves multiple genes that are transcriptionally regulated in response to injury or mitogenic signals implicating a higher level of regulation exists in VSMC that controls entire gene expression program required for proliferation [7,8] This regulation involves epigenetic mechanisms, which by altering the chromatin structure and dynamics control transcriptional gene activation that are essential for normal development and maintenance of organisms and to facilitate their interaction with surrounding environment [3,4,9,10,11]. Most HDACi exhibit multiple cellular effects, which are linked to chromatin-mediated altered transcriptional activity They inhibit cell proliferation, stimulate cell differentiation and/or induce apoptosis/cell death by selectively modulating gene expression [13,16,17]. Effects of butyrate and TSA on VSMC is investigated to determine whether upregulation of cyclin D1 in VSMC is restricted to butyrate alone or common to other HDACi, and to understand the mechanism and significance of cyclin D1upregulation in proliferation arrested VSMC
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