Cellular Effects of Butyrate on Vascular Smooth Muscle Cells are Mediated through Disparate Actions on Dual Targets, Histone Deacetylase (HDAC) Activity and PI3K/Akt Signaling Network

Abstract

Vascular remodeling is a characteristic feature of cardiovascular diseases. Altered cellular processes of vascular smooth muscle cells (VSMCs) is a crucial component in vascular remodeling. Histone deacetylase inhibitor (HDACI), butyrate, arrests VSMC proliferation and promotes cell growth. The objective of the study is to determine the mechanism of butyrate-induced VSMC growth. Using proliferating VSMCs exposed to 5 mM butyrate, immunoblotting studies are performed to determine whether PI3K/Akt pathway that regulates different cellular effects is a target of butyrate-induced VSMC growth. Butyrate inhibits phosphorylation-dependent activation of PI3K, PDK1, and Akt, eliciting differential effects on downstream targets of Akt. Along with previously reported Ser9 phosphorylation-mediated GSK3 inactivation leading to stability, increased expression and accumulation of cyclin D1, and epigenetic histone modifications, inactivation of Akt by butyrate results in: transcriptional activation of FOXO1 and FOXO3 promoting G1 arrest through p21Cip1/Waf1 and p15INK4B upregulation; inactivation of mTOR inhibiting activation of its targets p70S6K and 4E-BP1 impeding protein synthesis; inhibition of caspase 3 cleavage and downregulation of PARP preventing apoptosis. Our findings imply butyrate abrogates Akt activation, causing differential effects on Akt targets promoting convergence of cross-talk between their complimentary actions leading to VSMC growth by arresting proliferation and inhibiting apoptosis through its effect on dual targets, HDAC activity and PI3K/Akt pathway network.

Keywords: Akt; butyrate; histone deacetylase inhibitor; signaling; vascular smooth muscle cells.

Figure 1

Cellular effects of butyrate on VSMCs. Proliferating VSMCs treated with (BA) or without (Con) 5 mM butyrate for 72 h and processed for assessing butyrate-induced cellular effects. (A) Pseudocolored phase contrast images and corresponding nuclear images of VSMCs stained with Hoechst reveal butyrate-induced altered cell morphology and increase in size. The scale bar is 50 μm. (B) VSMC proliferation measured by cell counting indicate proliferation arrest by butyrate. ** p < 0.001 vs Control (Con).

Figure 2

Effect of butyrate (BA) on PI3K in VSMCs. Proliferating VSMCs were treated with or without 5 mM butyrate for indicated periods of time. At the end of treatment, cell lysates were prepared and subjected to Western blot analysis to determine the PI3K expression and activation level by measuring the level of unphosphorylated and phosphorylated p85 subunit of PI3K, respectively. Immunoblotting of ERK1/2 was performed with the same lysate to normalize the protein loading. The band intensities were measured and normalized to protein loading. The data obtained were analyzed and presented as mean ± S.D. (A) Expression level of PI3K p85 subunit measured by using antibody specific to p85 subunit (* p < 0.01 vs 24 h control (Con), and ** p < 0.001 vs 30 h control (Con). (B) Activation of p85 subunit of PI3K was evaluated by the antibody specific to Tyr458-phosphorylated p85 (** p< 0.001 vs respective control (Con).

Figure 3

Effect of butyrate on PDK1 protein and phosphorylation level in VSMCs. Proliferating VSMCs were treated with (BA) or without (Con) butyrate for indicated periods of time and then cell lysates were prepared. Cell lysates were processed for PDK1 Western blotting to assess the effect of butyrate-inhibited PI3K activity on PDK1 protein and activation levels by measuring total and phosphorylated PDK1 levels, respectively. The data obtained were analyzed and presented as mean ± S.D. (A) PDK1 protein level measured by using antibody specific to total PDK1 (B) Phosphorylation of PDK1 assessed by using antibody specific to Ser241-phosphorylated PDK1 indicated significant reduction in activation of PDK1 in VSMCs treated with butyrate for 24 h and 30 h (* p < 0.01 vs 24 h control (Con), ** p < 0.001 vs 30 h control (Con).

Figure 4

Inhibition of Akt activation by butyrate. VSMCs were treated with (BA) or without (Con) butyrate for indicated periods of time and then processed for western analysis of total Akt, Thr308 and Ser473 phosphorylated Akt as described in Methods Section. Total Akt levels were determined by immunoblotting with Akt-specific antibody and used as normalizing controls. Respective data are presented as mean ± S.D. (A) Levels of Thr308-phosphorylated Akt were determined by immunoblotting with antibody specific to phospho-Thr308Akt. (B) Levels of Ser473-phosphorylated Akt were assessed by immunoblotting with antibody specific to phospho-Ser473Akt. ^℗℗ p < 0.001 vs 6 h control (Con); ** p< 0.001 vs 6 h control (Con), * p < 0.01 vs 24 h control (Con).

Figure 5

Evaluation of butyrate influence on phosphorylation state of FOXO1. VSMCs treated with (BA) or without (Con) butyrate for indicated periods were processed for Western blot analysis of phosphorylated FOXO1 at Ser256 and Ser329. (A) Phosphorylation state of FOXO1 at Ser256 is assessed by immunoblotting with antibody specific for FOXO1 phosphorylated at Ser256. (^℗℗ p < 0.001 vs 6 h control) and is notably reduced in butyrate-treated VSMCs at 24 h and 30 h (** p < 0.001 vs respective control). (B) Phosphorylation of FOXO1 at Ser329 is determined by the antibody specific to FOXO1 phosphorylated at Ser329. (** p <0.001 vs respective control).

Figure 6

Assessment of butyrate effect on FOXO3. Cell lysates prepared from VSMCs treated with (BA) or without (Con) butyrate for indicated periods were subjected to Western blot analysis. (A) Analysis of total FOXO3 by immunoblotting with antibody specific to FOXO3. (B) Phosphorylation of FOXO3 at Ser253.* p< 0.01 vs 6 h control (Con); ** p< 0.001 vs respective control (Con). (C) Phosphorylation of FOXO3 at Ser318/321. ^℗ p< 0.01 vs 6 h control (Con); ** p< 0.001 vs respective control (Con).

Figure 7

Evaluation of butyrate effect on caspase 3 and PARP in VSMCs. Cell lysates prepared from VSMCs treated with (BA) or without (Con) butyrate were processed for Western blot analyses using antibodies specific to respective proteins. (A) Caspase 3 exhibits no activation both in control and butyrate-treated VSMCs except for trace level of cleaved caspase 3 in VSMCs treated with butyrate for 30 h (B) Full length PARP was determined by immunoblotting with antibody specific to full length PARP (^@@ p < 0.001 vs 6 h control and ** p < 0.001 vs respective control). (C) Cleaved PARP was assessed by probing with antibody specific to cleaved PARP (^@@ p < 0.001 vs 6 h control but no cleaved PARP was detected in treated VSMCs (** p < 0.001 vs respective control).

Figure 8

Inhibition of activation of mTOR and its downstream targets by butyrate in VSMCs. Cell lysates prepared from VSMCs treated with (BA) or without (Con) butyrate were analyzed by Western blotting using antibodies specific to (A) phosphorylated mTOR, (B) Phosphorylated p70S6 kinase, (C) phosphorylated S6 ribosomal protein and (D) phosphorylated 4E-BP1. Appropriate second antibodies labelled with IRDye 680LT or IRDye 800CW were used for detecting target proteins. LI-COR CLx imaging system and Image Studio software were used for scanning and quantitating signal intensities. (* p < 0.01 vs Control).

Figure 9

Butyrate-inhibited Akt activation arrests VSMC proliferation promoting VSMC growth via its downstream targets. Increased expression of cyclin D1, and its stability due to inhibition of GSK3 activity along with increased expression of p21Cip1/Waf1 and p15INK4B through the activation of FOXO proteins by butyrate arrests VSMCs in G1-phase (red arrows). Additionally, inhibition of caspase 3 activity and downregulation of PARP by butyrate inhibits apoptosis. Inhibition of mTOR-mediated protein synthesis appears to have no effect on VSMC growth.