In earlier studies of EC migration, we showed that a lysoPC-induced rise in [Ca2+
caused cytoskeletal changes and inhibition of movement, but the ion channels responsible were not identified (Chaudhuri et al., 2003
). The present studies show that lysoPC activates endogenous TRPC channels in ECs. TRPC5 and TRPC6 are externalized in the plasma membrane with subsequent increased [Ca2+
and disruption of EC migration. TRPC6 is involved in vascular endothelial growth factor–mediated increase in vascular permeability and in EC contraction in response to thrombin (Pocock et al., 2004
; Singh et al., 2007
), but other functions of TRPC5 and TRPC6 in ECs are largely unknown. The role of TRPC5 or TRPC6 in the regulation of EC migration has not been reported previously, and our data suggest that a TRPC6-TRPC5 activation cascade is a critical element in the inhibition of EC migration by lysoPC.
Events associated with the activation of TRPC channels include externalization and ion flux. In HEK-293 cells overexpressing TRPC5, growth factors cause the rapid translocation of TRPC5 from vesicles to the plasma membrane and increase functional TRPC5 current (Bezzerides et al., 2004
). Serum growth factors in EC tissue culture did not appear to influence translocation of TRPC5 and TRPC6 in our studies. Baseline localization and lysoPC-induced translocation was similar in ECs made quiescent in serum-free medium and in those maintained in 10% FBS up to initiation of experiments. TRPC6 proteins are localized in the caveolae-related microdomain vesicles, and during activation these vesicles fuse to the plasma membrane to externalize TRPC6 (Cayouette et al., 2004
). Tyrosine phosphorylation regulates TRPC6 activity (Hisatsune et al., 2004
), and increases membrane insertion of TRPC4 (Odell et al., 2005
), but it is not clear whether tyrosine phosphorylation is required for channel protein externalization, modification of activity, or channel opening. LysoPC induces tyrosine phosphorylation of TRPC5 and TRPC6 (), and general tyrosine kinase inhibitors inhibit TRPC6 externalization (data not shown), suggesting that tyrosine phosphorylation is required for externalization. Although, fyn and src tyrosine kinases interact with TRPC6 to increase TRPC6 activation in COS-7 cells (Hisatsune et al., 2004
), the specific kinase responsible for lysoPC-induced tyrosine phosphorylation of TRPC6 in ECs has not been identified.
LysoPC activates TRPC5. In HEK-293 cells overexpressing TRPC5, lysoPC activates TRPC5, and this effect is seen even in excised membrane patches leading investigators to conclude that lysoPC has a relatively direct effect on the channel (Flemming et al., 2006
). Our studies suggest that in aortic ECs with only endogenous TRPC proteins, lysoPC-induced TRPC6 activation precedes and contributes to TRPC5 translocation. Down-regulation of TRPC6 in ECs inhibits lysoPC-induced TRPC5 externalization. TRPC5 can be activated by various pathways including receptor activation, external ionic activation, increased [Ca2+
, and store-operated mechanisms (Zeng et al., 2004
). We postulate that the rise in [Ca2+
through TRPC6 channels activates TRPC5. Increased [Ca2+
can induce calcium-dependent signaling events such as myosin light-chain kinase activation that can activate TRPC5 (Shimizu et al., 2006
). The role of myosin light-chain kinase in lysoPC-induced activation of TRPC5 is currently under investigation in our laboratory. Interestingly, the early rise in [Ca2+
persists when TRPC5 is down-regulated using siRNA, consistent with lysoPC-induced opening of a calcium channel, such as TRPC6, and subsequent Ca2+
entry. In the absence of extracellular calcium, lysoPC induces the translocation of TRPC6 (B), but [Ca2+
does not increase (Chaudhuri et al., 2003
), and TRPC5 is not externalized (B). Furthermore, lysoPC does not induce TRPC5 translocation in EC preincubated with intracellular calcium chelators, BAPTA-AM or EGTA-AM, again suggesting that a [Ca2+
rise is required for activation of TRPC5 by lysoPC. The activation of TRPC5 is not simply a response to any increase in [Ca2+
because the increase in [Ca2+
secondary to bradykinin does not activate TRPC5 (data not shown). We postulate that the increased [Ca2+
must occur within specific spatial boundaries in the cell to activate TRPC5, suggesting that TRPC5 and TRPC6 are functionally connected for this unique activation cascade to occur.
Functional TRPC channels are thought to be tetramers that can be homotetramers or heterotetramers (Hofmann et al., 2002
). In general, endogenously expressed TRPC proteins form heteromultimers composed of members from the same subgroup (Goel et al., 2002
). TRPC6 and TRPC5 belong to different TRPC subgroups and are unlikely to coassemble. TRPC channels from the different subgroups can form heteromers under specific circumstances. TRPC3 and TRPC6 can form a heteromeric channel complex with TRPC1, TRPC4, and TRPC5 in rat embryonic brain but not in adult brain (Strübing et al., 2003
). Our coimmunoprecipitation studies suggest that TRPC5 and TRPC6 are not coassembled in a heteromeric channel (data not shown), in agreement with a previously published report (Goel et al., 2005
). TRPC6 and TRPC5 protein could form heteromultimers that are not identified in coimmunoprecipitation studies due to low channel density or antigenic sites hidden by heteromultimer formation. However, the correlation of TRPC6 activation with initial increase of calcium and the lag in TRPC5 activation argue against a TRPC5-TRPC6 heteromultimer and instead suggest a novel TRPC6-initiated, functional TRPC6-TRPC5 channel cascade.
Activation of a novel TRPC6-TRPC5 channel cascade plays a key role in calcium entry and inhibition of EC migration by lysoPC. A spike in [Ca2+
is needed to initiate cell movement (Tran et al., 1999
), but a prolonged increase, as is seen in ECs incubated in the presence of lysoPC, inhibits EC migration. Our data suggest that lysoPC initially activates TRPC6, causing increased [Ca2+
that leads to externalization of TRPC5, which allows a prolonged increase in [Ca2+
that inhibits EC migration. Down-regulation of TRPC6 inhibits TRPC5 activation, suggesting a TRPC6-dependent activation of TRPC5 by lysoPC. Although increased [Ca2+
is only one of several pathways by which lysoPC inhibits EC migration (Ghosh et al., 2002
; Chaudhuri et al., 2005
), partial preservation of migration is achieved by preventing the lysoPC-induced [Ca2+
rise. A better understanding of the TRPC6-TRPC5 activation cascade will allow development of therapeutic agents to preserve EC movement and promote angiogenesis and healing of EC injuries in atherosclerotic arteries.