No abstract

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30-50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, ␣ v ␤ 3 integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with ␣ v ␤ 3 integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or ␣ v ␤ 3 integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-␣ v ␤ 3 interaction was blocked by treatment of CASMCs with antibodies against OPN or ␣ v ␤ 3 integrin. Our results demonstrate that OPN and ␣ v ␤ 3 integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-␣ v ␤ 3 interaction may provide a therapeutic approach to preventing restenosis.

The ability of cAMP response-element binding protein (CREB)-binding protein (CBP) to function as a co-activator for a number of transcription factors appears to be mediated by its ability to act as a histone acetyltransferase and through its interaction with a number of other proteins (general transcription factors, histone acetyltransferases, and other co-activators). Here we report that CBP also interacts with a novel ATPase termed Snf2-Related CBP Activator Protein (SRCAP). Consistent with this activity, SRCAP contains the conserved ATPase domain found within members of the Snf2 family. Transfection experiments demonstrate that SRCAP is able to activate transcription when expressed as a Gal-SRCAP chimera and that SRCAP also enhances the ability of CBP to activate transcription. The adenoviral protein E1A was found to disrupt interaction between SRCAP and CBP possibly representing a mechanism for E1A-mediated transcriptional repression. CREB 1 -binding protein (CBP) has been found to function as a co-activator for a growing number of sequence specific transcription factors including CREB, the STATs, and the nuclear receptors (1-5). Binding studies have identified several regions of CBP that interact with general transcription factors such as TBP, TFIIB, and RNAP II (2, 6 -8), suggesting it functions as a co-activator in part by recruiting these proteins to the promoter. CBP has also been shown to have intrinsic histone acetyltransferase (HAT) activity and to bind to several proteins with HAT activity (P/CAF, ACTR, NCoA-1). This suggests that CBP alone, or acting in conjunction with these proteins, functions as a co-activator by stimulating remodeling of chromatin (9 -12). This is supported by the work of Korus et al. (13) who demonstrate that several transcription factors have a specific requirement for the HAT activity of NCoAs, P/CAF, and CBP for activation of transcription. The adenoviral protein E1A also binds CBP but represses the ability of CBP to function as a co-activator for CREB as well as a number of other transcription factors (4,5,14,15). This appears to be due in part to the ability of E1A to prevent binding of P/CAF and P/CIP to the C-terminal end of CBP. E1A also binds to the N-terminal end of CBP and suppresses the ability of a Gal-CBP-(1-450) chimera to activate transcription. Although P/CAF also binds to this same region, competition between P/CAF and E1A binding has not been demonstrated (5).Deletion of amino acids 1-460 abolishes the ability of CBP to serve as a co-activator for CREB and STAT-1 but not for other transcription factors such as the retinoic acid receptor (5, 6). In "squelching-type" assays, overexpression of CBP amino acids 1-460 has also been found to block the ability of full-length CBP to activate CREB-mediated transcription (5). Studies from several laboratories indicate that this region of CBP contacts proteins, including TBP and P/CAF, which may be involved in the activation of transcription (5, 6). Microinjection studies support such a role for P/CAF by demonstrating ...

The tyrosine kinasee activity of p60c-src, the protein product of the c-src gene, increases during mitosis; this may be important in initiating at least some of the cellular changes that occur during this phase of the cell cycle. Although there is evidence that p60c-src is phosphorylated at several sites during mitosis, phosphorylation in vitro does not increase its kinase activity. We now report that the kinase activity of a p60c-src mutant with residue tyrosine 527 changed to phenylanine does not change during the cell cycle, suggesting that changes in the phosphorylation state of this residue may be responsible for the activation of p60c-src at mitosis. Although changes in phosphorylation at Tyr 527 cannot be detected with the wild-type protein we find that phosphorylation at Tyr 527 of a mutant with reduced kinase activity decreases threefold during mitosis. On the basis of these results we suggest that activation of p60c-src at mitosis results from decreased phosphorylation on Tyr 527, and that p60c-src may be or may activate the kinase that phosphorylates Tyr 527.

Phosphorylation of pp6wcsrc by p34c2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosisspecific decrease in Tyr-527 phosphorylation and half the increase in pp60-src kinase activity. We conclude that p34cdc2 partially activates pp6Wcsrc by a two-step process in which its serine/threonine phosphorylations either sensitize pp6Oe-sr' to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase.Furthermore, additional events, independent of these p34-2 mediated phosphorylations, participate in mitotic activation of pp6Ocerc.p34cdc2, a critical cell cycle regulator (see refs. 1 and 2 for reviews), is activated at the onset of mitosis as a serine/ threonine protein kinase by an intricate sequence of dephosphorylation and phosphorylation events and by its association with cyclin proteins. Activated p34cdc2 is thought to initiate a cascade of protein kinases and, possibly, phosphatases that results in mitosis-specific cytostructural rearrangements such as cell rounding, nuclear envelope breakdown, and mitotic spindle formation. p34cdc2 phosphorylates chicken pp60c-src, the product of the chicken c-src protooncogene, in vitro at residues . These same sites are phosphorylated during mitosis in genetically modified NIH 3T3 mouse fibroblasts that overexpress chicken pp60c-src (3, 4). Thr-34, Ser-72, and their flanking p34cdc2 substrate-consensus sequences are conserved in frog, chicken, mouse, and human pp60c-src (5-8) and are phosphorylated during mitosis at least in mouse (unpublished results) and human (9) pp60c-src. The mitosisspecific phosphorylations (MSPs) ofpp60c-src in cultured cells retard its electrophoretic mobility and are associated with a 2-to 9-fold increase in specific kinase activity (4, 10,11). Both these mitosis-specific effects disappear as fibroblasts exit mitosis (4).These facts suggest that pp60c-src may participate in the transduction of p34cdc2-initiated signals and mitotic protein kinase cascades (see ref. 12 for review). However, phosphorylation of pp60c-src by p34cdc2 does not stimulate its specific kinase activity (3,9). This suggests that some other mitosisspecific event is involved in the activation. This event appears to be partial dephosphorylation of Tyr-527 (10, 11), a residue that is highly (>90%) phosphorylated in wild-type (wt) pp60c-src in unsynchronized cells (13). Complete inhibition of Tyr-527 phosphorylation causes a 10-to 30-fold stimulation of pp60c-src specific kinase activity (see ref. 14 for review), so the 2-to 3-fold mitotic increase in kinase activity observed in our system could be caused by a 10-20o decrease in Tyr-527 phosphorylation. The small magnitude of this predicted change has preclude...