Induction of Smooth Muscle α-Actin in Vascular Smooth Muscle Cells by Arginine Vasopressin Is Mediated by c-Jun Amino-terminal Kinases and p38 Mitogen-activated Protein Kinase

Abstract

Exposure of vascular smooth muscle cells to arginine vasopressin (AVP) increases smooth muscle ␣-actin (SM-␣-actin) expression through activation of the SM-␣-actin promoter. The goal of this study was to determine the role of the mitogen-activated protein kinase (MAP kinase) family in regulation of SM-␣-actin expression. AVP activated all three MAP kinase family members: ERKs, JNKs, and p38 MAP kinase. Inhibition of JNKs or p38 decreased AVP-stimulated SM-␣-actin promoter activity, whereas inhibition of ERKs had no effect. A 150-base pair region of the promoter containing two CArG boxes was sufficient to mediate regulation by vasoconstrictors. Mutations in either CArG box decreased AVPstimulated promoter activity. Electrophoretic mobility shift assays using oligonucleotides corresponding to either CArG box resulted in a complex of similar mobility whose intensity was increased by AVP. Antibodies against serum response factor (SRF) completely supershifted this complex, indicating that SRF binds to both CArG boxes. Overexpression of SRF increased basal promoter activity, but activity was still stimulated by AVP. AVP stimulation rapidly increased SRF phosphorylation. These data indicate that both JNKs and p38 participate in regulation of SM-␣-actin expression. SRF, which binds to two critical CArG boxes in the promoter, represents a potential target of these kinases.

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).…”

“…Affymetrix Microarray Analysis-VSMC stimulated with AVP or PDGF for 6 or 24 h and VSMC stably expressing H-Ras (14), Myr-Akt (15), or G␣ 16 (12) along with their control cells were subjected to total RNA isolation by using RNeasy miniprep (Qiagen). Labeling of the probes for microarray was performed as described previously (27).…”

“…Cells were incubated for 16 h in Eagle's minimal essential medium with 10% fetal calf serum and then placed in Eagle's minimal essential medium with 0.2% fetal calf serum with or without agonists for 72 h. The transiently transfected VSMC were then washed twice with ice-cold phosphate-buffered saline and harvested in luciferase reporter lysis buffer (Promega, Madison, WI). Cell lysates were centrifuged, and the supernatants were assayed for luciferase and ␤-galactosidase activities as previously described (16).…”

“…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.…”

Transcriptional Regulation of a Contractile Gene by Mechanical Forces Applied through Integrins in Osteoblasts

“…However, the force-induced activation of the SRE in the SMA promoter in connective tissue cells such as osteoblasts may be different from the mechanisms involved in c-Fos activation and may also be cell type-specific. Notably, applied mechanical force can regulate SMA gene expression by the p38 kinase pathway in cardiac fibroblasts (22), a process that in turn may regulate the binding of the serum response factor (SRF) to two critical CArG boxes in the SMA promoter (23). Cognizant of this background we have examined the responsiveness of the SMA promoter to applied forces in an in vitro model system (24,25).…”