Our previous studies have demonstrated that both the RhoA/
Rho kinase and the
protein kinase C (PKC) signaling pathways are involved in the low-dose
endothelial monocyte-activating polypeptide-II (
EMAP-II)-induced blood-
tumor barrier (BTB) opening. In the present study, an in vitro BTB model was used to investigate which
isoforms of PKC were involved in this process as well as the interactions between the RhoA/
Rho kinase and the PKC signaling pathways. Our results showed that
EMAP-II-activated PKC-α, β, and ζ and induced translocations of them from the cytosolic to the membrane fractions of rat brain microvascular endothelial cells. The
EMAP-II-induced alterations in BTB permeability and tight junction (TJ)
protein expression were partially blocked by GÖ6976, the inhibitor of PKC-α/β, and PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI). Meanwhile, we observed that GÖ6976 partly inhibited the
EMAP-II-induced rearrangement of actin cytoskeleton as well as phosphorylation of
myosin light chain and
cofilin, whereas PKC-ζ-PI had no effect on these above-mentioned changes induced by
EMAP-II. Also, our data revealed that inhibition of RhoA or inhibition of
Rho kinase significantly diminished the activities and the translocations of PKC-α and PKC-β induced by
EMAP-II, whereas PKC-ζ was unaffected. However, inhibition of PKC-α/β or inhibition of PKC-ζ did not cause any changes in the RhoA and
Rho kinase activities. The effects of
EMAP-II on BTB permeability and TJ
proteins expression were completely blocked by inhibition of both RhoA and PKC-ζ, whereas inhibition of both RhoA and PKC-α/β had an effect similar to that of inhibition of RhoA alone. In summary, this study demonstrates for the first time that three PKC
isoforms, PKC-α, β, and ζ, are involved in the
EMAP-II-induced BTB opening. It is PKC-α/β, but not PKC-ζ, which serves as the downstream target for RhoA and
Rho kinase, suggesting that
EMAP-II induces BTB opening via the RhoA/
Rho kinase/PKC-α/β signaling pathways. However, PKC-ζ is involved in this process by other mechanisms.