Phenylephrine induces elevated RhoA activation and smooth muscle alpha-actin expression in Pkd2+/- vascular smooth muscle cells.
The mechanisms underlying vascular complications in autosomal-dominant polycystic kidney disease (ADPKD) have not been fully elucidated. However, molecular components altered in Pkd mutant vascular smooth muscle cells (VSMCs) are gradually being identified. Pkd2(+/-) arterial smooth muscles show elevated levels of (1) phenylephrine (PE)-induced, Ca(2+)-independent vasocontraction and (2) smooth muscle alpha-actin (SMA) expression. As these two processes are heavily influenced by RhoA signaling and by cellular filamentous-to-globular (F/G)-actin dynamics, we examined PE-induced changes in RhoA activation and the F/G-SMA ratio in wild-type (wt) and Pkd2(+/-) VSMCs; we further tested the hypothesis that the abnormal response to PE and the resultant elevation in the F/G-SMA ratio contribute to the exuberant SMA expression in Pkd2(+/-) VSMCs. GTP-RhoA and F/G-SMA in mouse aortic media and primary cultured VSMCs were determined using RhoA activation and in vivo F-to-G-actin assays. Myocardin-related transcription factor-A (MRTF-A) (SMA transcription coactivator) was localized by immunofluorescence, nuclear MRTF-A quantified by western analysis using nuclear extracts and SMA expression by luciferase reporter assay. PE induced a >3-fold higher RhoA activation in Pkd2(+/-) than in wt VSMCs and higher levels of downstream p-LIMK and p-cofilin. Moreover, Pkd2(+/-) VSMCs showed a higher baseline and PE-induced F/G-SMA ratio. The F/G-SMA elevation enhanced nuclear translocation of MRTF-A, which upregulated SMA transcription. In summary, PE-induced RhoA hyperactivation and defects in F-to-G SMA balance likely have a role in the abnormal vasocontraction and SMA expression in Pkd2(+/-) arteries. These defects could potentially contribute to the genesis of vascular complications in ADPKD, thus providing new areas for further research and therapeutic targeting.
Du
Hui
H
Department of Medicine, Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, Mayo Graduate School, Rochester, MN 55905, USA.
Wang
Xiangling
X
Wu
Jun
J
Qian
Qi
Q
D000199:Actins / Q000096:biosynthesis*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology*; D050396:TRPP Cation Channels / Q000096:biosynthesis* / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D009687:Nuclear Proteins; D010656:Phenylephrine; D050396:TRPP Cation Channels; D015534:Trans-Activators; D014662:Vasoconstrictor Agents; D020742:rhoA GTP-Binding Protein; C511173:Mrtfa protein, mouse