Essential roles of IkappaB kinases alpha and beta in serum- and IL-1-induced human VSMC proliferation.

Interleukin-1 (IL-1) is a potent vascular smooth muscle cell (VSMC) mitogen, which can stimulate cells via activation of nuclear factor-kappaB (NF-kappaB) following phosphorylation of its inhibitory subunit (IkappaB). Because the proliferative effect of IL-1 is additive with that of serum, the present studies assessed the role of IkappaB kinases (IKKs) and NF-kappaB in both IL-1- and serum-induced VSMC proliferation. IL-1beta (1 ng/ml) induced marked and persistent NF-kappaB activation in VSMC that was maximal at 1 h and persisted for 3 days. There was a 3-fold increase in DNA synthesis after acute IL-1 exposure (24-96 h) and a 12-fold increase after chronic IL-1 exposure (>7 days). Electrophoretic mobility shift assay and supershift analysis indicated that IL-1-induced NF-kappaB complexes consisted of p65/p50 heterodimers and p50 homodimers. Human saphenous vein smooth muscle cells (HSVSMC) were transiently cotransfected with expression plasmids encoding a dominant negative mutant form of either IKKalpha or IKKbeta, in which K(44) was mutated to A (K44A), and a green fluorescent protein expression plasmid that allows identification of transfected cells. IL-1 induced nuclear localization of p65 in 95% of cells transfected with vector alone but in only 69% and 26% of cells expressing IKKalpha (K44A) or IKKbeta (K44A), respectively. Likewise, proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been transfected with vector alone, but only 2.2- and 1.5-fold proliferation in HSVSMC expressing IKKalpha (K44A) or IKKbeta (K44A), respectively. Although serum activated NF-kappaB transiently, serum-induced proliferation was markedly attenuated in HSVSMC expressing IKKalpha (K44A) and IKKbeta (K44A) compared with HSVSMC transfected with vector alone. The results support an essential role of IKKs in the proliferative response of HSVSMC to IL-1 and to serum.