Platelet-derived Growth Factor-BB-mediated Activation of Akt Suppresses Smooth Muscle-specific Gene Expression through Inhibition of Mitogen-activated Protein Kinase and Redistribution of Serum Response Factor

Abstract

Platelet-derived growth factor (PDGF) inhibits expression of smooth muscle (SM) genes in vascular smooth muscle cells and blocks induction by arginine vasopressin (AVP). We have previously demonstrated that suppression of SM-␣-actin by PDGF-BB is mediated in part through a Ras-dependent pathway. This study examined the role of phosphatidylinositol 3-kinase (PI3K)y and its downstream effector, Akt, in regulating SM gene expression. PDGF caused a rapid sustained activation of Akt, whereas AVP caused only a small transient increase. PDGF selectively caused a sustained stimulation of p85/p110␣ PI3K. In contrast, p85/110␤ PI3K activity was not altered by either PDGF or AVP, whereas both agents caused a delayed activation of Class IB p101/110␥ PI3K. Expression of a gain-of-function PI3K or myristoylated Akt (myr-Akt) mimicked the inhibitory effect of PDGF on SM-␣-actin and SM22␣ expression. Pretreatment with LY 294002 reversed the inhibitory effect of PDGF. Expression of myr-Akt selectively inhibited AVP-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, which we have shown are critical for induction of these genes. Nuclear extracts from PDGF-stimulated or myr-Akt expressing cells showed reduced serum response factor binding to SM-specific CArG elements. This was associated with appearance of serum response factor in the cytoplasm. These data indicate that activation of p85/p110␣/Akt mediates suppression of SM gene expression by PDGF.

Insulin attenuates vascular smooth muscle calcification but increases vascular smooth muscle cell phosphate transport

“…The growth medium does not contain insulin; relevant molecules include calcium (1.8 mM), phosphate (1.014 mM), and glucose (5.6 mM). VSMC primary cultures were used up to the ninth passage (14). All cells were placed in serum-reduced medium (≤0.2% serum) for 24 hr to maintain quiescence prior to further treatment.…”

Patterns of Gene Expression Differentially Regulated by Platelet-derived Growth Factor and Hypertrophic Stimuli in Vascular Smooth Muscle Cells

“…Cells (passages 4 -12) were grown in Eagle's minimal essential medium containing 1 mM L-glutamine, 2 g/liter NaHCO 3 , 100 international units/ml penicillin, 100 g/ml streptomycin, and 10% (v/v) fetal calf serum at 37°C in a humidified air/CO 2 (19:1) atmosphere. For the stable transfections of VSMC with H-Ras, Myr-Akt, and G␣ 16 , cDNAs encoding either Akt containing the Src myristoylation sequence (15), the GTPase-deficient, constitutively active forms of the human T24 H-Ras with V12 (G12V) mutation (14), or GTPase-deficient G␣ 16 (␣ 16 Q212L) (12) were packaged into a replication-defective retrovirus by the procedure described previously (26). Secreted retrovirus was supplemented with Polybrene (8 g/ml), filtered (0.45 m), and incubated with VSMC for 48 h. Cells expressing myristoylated Akt, H-Ras, or G␣ 16 were selected by culturing them in medium containing G418 (500 g/ml).…”

“…VSMC were stimulated with PDGF-BB for 6 or 24 h, and gene expression was compared with control cells Tables A and B). Our laboratory previously showed that PDGF-BB-mediated suppression of SM-specific gene expression is partly mediated by activation of both the H-Ras and phosphatidylinositol 3-kinase/Akt pathways (14,15). To investigate the contribution of these pathways to the changes in the global gene expression profile induced by PDGF, we compared the increases and decreases induced by PDGF stimulation to the changes seen in VSMC stably expressing H-Ras (14) or constitutively active Myr-Akt (15) (see Supplementary Tables A and B).…”

“…In contrast, vasoconstrictors such as vasopressin (AVP) and angiotensin II promote increased contractility and induce hypertrophy (10 -12). Previous studies from our laboratory have demonstrated that these two classes of agents have opposing effects on expression of smooth muscle markers (SM markers), specifically contractile proteins such as smooth muscle ␣-actin and SM22␣ (13)(14)(15), with AVP increasing and PDGF suppressing transcription of these genes.…”

“…PDGF-induced suppression is mediated through activation of Ras and phosphatidylinositol 3-kinase/Akt pathways (14,15), whereas AVP-mediated regulation of SM markers involves activation of heterotrimeric G proteins of the G q family, leading to stimulation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinases (12,16). However, the phenotypic changes mediated by these agents are probably not exclusively mediated through changes in expression of contractile proteins but will instead be a consequence of more global changes in gene expression.…”