Mechanism Of SM22α Regulating GLUT4 Translocation And The Significance In VSMC Proliferation

Restenosis after balloon angioplasty and atherosclerosis are more common in individuals with diabetes than in the general population. Vascular smooth muscle cell(VSMC) proliferation consumes glucose for metabolic purposes. Increased glucose transport, glycolytic flux and mitochondrial oxygen consumption are thus critical features in the bioenergetic shift that occurs during VSMC proliferation.The insulin-sensitive glucose transporter 4(GLUT4) participates in glucose transport in VSMCs and is significantly upregulated in the neointima. Platelet-derived growth factor(PDGF) facilitates glucose uptake by inducing GLUT4 translocation via cellular cortical actin remodeling.Smooth muscle(SM) 22α, an actin-binding protein, is involved in actin filament assembly and cytoskeletal rearrangements of VSMCs. The expression of SM22α is downregulated in proliferation-related diseases such as atherosclerotic plaques, abdominal aortic aneurysms, and several types of cancer. We have previously demonstrated that high expression of SM22α inhibits VSMC proliferation and neointimal formation via blockage of the Ras-ERK1/2 signaling pathway activated by PDGF-BB, while SM22α disruption results in actin cytoskeleton remodeling similar to that observed following PDGF-BB stimulation. However, it is unknown whether SM22α is involved in PDGF-stimulated GLUT4 translocation. In the present study, we focused on the cellular mechanism and significance of PDGF-BB-induced GLUT4 translocation in VSMCs, and examined the effects of SM22α on this process.Methods:We measured PDGF-BB-induced GLUT4 translocation in VSMCs, and examined the effects of actin dynamics using immunofluorescence and western blot. The glucose uptake in VSMCs and in the aortas was detected using 2-NBDG. SM22α si RNA was used to clarify the role of SM22α in PDGF-BB-induced GLUT4 translocation and glucose uptake. The cell counting and Brd U analysis were used to test the activity of the cell proliferation. A left carotid artery ligation model was established using WT and SM22α-/- mice and glucose content in injured carotids was measured by high-performance liquid chromatography(HPLC). The microtubule dynamics was detected using immunofluorescence.Results:1 SM22α is involved in PDGF-BB-induced GLUT4 translocation via modulation of actin dynamics1.1 PDGF-BB increases cell surface GLUT4 content and glucose uptake.VSMCs were exposed to PDGF-BB stimulation for 0, 5, 10, 15, 20 and 30 min, and membrane extracts were assessed by western blot analysis. The result revealed that PDGF-BB induced GLUT4 membrane translocation, which peaked at 15 min and declined within 30 min. Consistent with the time course profile of GLUT4 translocation, 2-NBDG uptake reached a maximum at 15 min after exposure to PDGF-BB, and then declined.1.2 PDGF-BB increases cell surface GLUT4 content via modulation of actin.The distribution of GLUT4 in the membrane region of VSMCs was examined using immunofluorescent staining. We showed that GLUT4 translocated from the cytosol to the cell periphery upon PDGF-BB stimulation, where it colocalized with polymerized cortical F-actin. However, pretreatment with JPK which stabilizes the actin cytoskeleton, abolished PDGF-BB-induced GLUT4 membrane translocation and decreased the cortical F-actin polymerization. Pretreatment with JPK significantly inhibit PDGF-BB-induced glucose uptake in VSMCs. These findings suggest that actin remodeling is required for PDGF-BB-induced GLUT4 translocation and glucose uptake in VSMCs.1.3 Down-regulation of SM22α increases PDGF-BB-induced GLUT4 translocation by promoting actin remodeling.SM22α-specific small interference RNA(si SM22α) was used to silence the expression of endogenous SM22α. We observed that knockdown of SM22α decreased the density of stress fibers, and displayed an increased cortical F-actin polymerization upon PDGF-BB stimulation. Meanwhile, knockdown of SM22α increased the distribution of GLUT4 in the membrane region upon PDGF-BB stimulation. This result was further verified by western blot. Upon PDGF-BB stimulation, VSMCs of Sm22α-/- mice displayed an increased cortical actin polymerization and GLUT4 accumulation in the cellular surface compared with that in WT mice. These findings support our hypothesis that the down-regulation of SM22α facilitates PDGF-BB-induced GLUT4 translocation by promoting actin dynamics and cortical F-actin polymerization.1.4 SM22α disruption promotes PDGF-BB-induced glucose uptake and metabolism.2-NBDG uptake was measured in SM22α-knockdown or Sm22α VSMCs. The results revealed that SM22α down-regulation or deletion led to an increase in PDGF-BB-induced 2-NBDG uptake, which was consistent with GLUT4 translocation under the same conditions. The increase in PDGF-BB-induced 2-NBDG uptake was inhibited by pretreatment with Indinavir that is an antagonist of GLUT4. We also observed that the activity of HK and LDH induced by PDGF-BB was increased by SM22α knockdown, suggesting that SM22α disruption accelerates energy metabolism in VSMCs.2 Deletion of SM22α promotes VSMC proliferation via promoting glucose uptake.2.1 GLUT4 expression and GLUT4-mediated glucose uptake are essential for VSMC proliferation.A left carotid artery ligation model was established using C57BL/6J WT mice. Immunofluorescence staining revealed that the expression of GLUT4 markedly increased with a decrease in α-actin expression level in neointima. In vitro studies, we found that PDGF-BB significantly elevated GLUT4 expression in VSMCs in a time-dependent manner, consistent with the expression of proliferating cell nuclear antigen(PCNA). Glucose consumption from the cell culture medium(low glucose DMEM) increased under the same conditions. Pre-incubation of VSMCs with the GLUT4 antagonist Indinavir resulted in significantly reduced glucose consumption, accompanied by decreased proliferation activity. The results suggested that GLUT4 expression and GLUT4-mediated glucose uptake stimulation are essential for VSMC proliferation induced by PDGF-BB.2.2 The disruption of SM22α improves 2-NBDG uptake in VSMCs.The carotid arteries of WT and Sm22α-/- mice were pre-incubated with or without Indinavir before exposing to PDGF-BB stimulation, the fluorescence intensity of 2-NBDG was analyzed. We found that 2-NBDG fluorescence intensity in Sm22α-/- carotid arteries increased compared with WT ones upon PDGF-BB stimulation, which were inhibited by Indinavir.2.3 GLUT4 contributes to neointimal hyperplasia in Sm22α-/- mice.To assess the significance of the increased glucose uptake observed in VSMCs, a left carotid artery ligation model was established using WT and Sm22α-/- mice. H&E staining revealed that the neointimal hyperplasia of Sm22α-/- mice was thicker than that of WT mice. Membrane extracts were assessed by western blot analysis. Compared with WT, GLUT4 expression in the membrane fractions of injured arteries from Sm22α-/- mice was increased. The glucose content of whole tissue in injured carotids from Sm22α-/- mice was significantly higher than those in the carotids of WT mice, which is consistent with increased neointimal thickness. These data suggest that glucose uptake induced by SM22α disruption may contribute to neointimal formation.3 SM22α increases VSMC microtubule stability.3.1 SM22α increases VSMC microtubule stability.Immunofluorescent staining with anti-α-tubulin antibody revealed that knockdown of SM22α induced the depolymerization of microtubules, which displayed unevenly aggregation around the nucleus. The thicker microtubule bundles with higher fluorescence intensity were found in SM22α-overexpressed cells. Similarly, the depolymerization of microtubules was found in Sm22α-/- VSMCs, which was abolished by the overexpression of SM22α. These results suggested that SM22α increases VSMC microtubule stability.3.2 SM22α increases the acetylation of the Lys40 residue in α-tubulin.Acetylation of α-tubulin Lys40 is regarded as a marker for evaluating stability of microtubules. We found that knockdown of SM22α decreased α-tubulin Lys40 acetylation. Conversely, overexpression of SM22α increased it.3.3 Deletion of SM22α reduces contact inhibition of proliferation, increases mitochondrial division and the mitochondrial membrane potential.Conclusions:1 PDGF-BB increases cell surface GLUT4 content and glucose uptake, contributing to VSMC proliferation.2 Deletion of SM22α promotes cortical F-actin polymerization, increases PDGF-BB-induced GLUT4 translocation, glucose uptake and metabolism in VSMCs.3 SM22α is a novel regulator of glucose metabolism associated with VSMC proliferation.4 SM22α promotes microtubule stability, contact inhibition of proliferation.