Class IIα
phosphoinositide 3-kinase is an essential regulator of
Ca2+-dependent
Rho GTPase activation, myosin phosphatase and contraction
in vascular smooth muscle cells
Kazuaki
Yoshioka, Noriko Takuwa, Naotoshi Sugimoto, and Yoh Takuwa
Department of Physiology, Kanazawa University Graduate
School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa
920-8640, Japan.
ABSTRACT
We previously demonstrated that phosphoinositide 3-kinase
(PI3K) inhibitors suppressed Rho activation and myosin
phosphatase (MLCP) inhibition in vascular smooth muscle
(VSM) strips, resulting in inhibition of 20-kDa myosin
light chain (MLC20)
phosphorylation and contraction. Based upon the analyses of
PI3K isoform expression in VSM and the compatibility of
PI3K inhibitor, we hypothesized that class II PI3K,
PI3K-C2, might be a responsible isoform that is involved in
the regulation of the Rho signaling pathway and contraction
in VSM. To test this hypothesis, we adopted a
differentiated VSM cell culture, which maintains a
contractile phenotype, and siRNA-mediated knockdown of
PI3K-C2 expression.
The siRNA-mediated knockdown of PI3K-C2α, but not class I
PI3K p110α, suppressed ionomycin-induced contraction
without altering Ca2+-mobilization.
Ionomycin-induced contraction was inhibited only by a
relatively high concentration of LY294002. Consistent with
our previous observations, ionomycin-induced contraction
was dependent on Rho and Rho-kinase in a
Ca2+-dependent
manner. Ionomycin induced phosphorylation of the
MLCP-regulatory subunit MYPT1 at Thr850/Thr695
and the MLC20
in a Rho kinase-dependent manner. Knockdown of PI3K-C2α
suppressed phosphorylation of both MYPT1 and MLC. The
receptor agonist noradrenaline, which induced a rapid
increase in the [Ca2+]i
and Ca2+-dependent
contraction, stimulated phosphorylation of MYPT1 and MLC in
a Ca2+-,
PI3K-C2α-, and Rho kinase-dependent manner. These
observations indicate that PI3K-C2α mediates
Ca2+-induced,
Rho- and Rho kinase-dependent negative regulation of MLCP,
and consequently MLC phosphorylation and contraction in
VSMCs.