Caveolin, a major
protein component of caveolae, directly interacts with multiple signaling molecules, such as Ras and
growth factor receptors, and inhibits their function. However, the role of the second messenger system in mediating this inhibition by
caveolin remains poorly understood. We examined the role of Ca2+-dependent signal in
caveolin- mediated growth inhibition using a rat cardiac myoblast cell line (H9C2), in which the expression of
caveolin- 3, the muscle specific subtype, can be induced using the LacSwitch system. Upon induction with
IPTG and serum-
starvation, the expression of
caveolin-3 was increased by 3.3-fold relative to that of mock-induced cells. The recombinant
caveolin-3 was localized to the same subcellular fraction as endogenous
caveolin-3 after
sucrose gradient purification.
Angiotensin II enhanced ERK phosphorylation, but this enhancement was significantly decreased in caveolin-3-induced cells in comparison to that in mock-induced cells. Similarly, when cells were stimulated with
fetal calf serum,
DNA synthesis, as determined by [3H]-
thymidine incorporation, was significantly decreased in
caveolin- 3-induced cells. When cells were treated with Ca2+
chelator (
BAPTA and
EGTA), however, this attenuation was blunted.
Calphostin (PKC inhibitor), but not
cyclosporine A treatment (
calcineurin inhibitor), blunted this attenuation in
caveolin-3 induced cells. Our findings suggest that
caveolin exhibits growth inhibition in a Ca2+-dependent manner, most likely through PKC, in cardiac myoblasts.