Transforming growth factor-beta 1 (TGF-beta 1) has been variably associated with the regulation of cellular proliferation and extracellular matrix expansion after arterial injury. We tested these associations in vivo in the rat carotid injury model. At 0, 3, 7, 14 and 28 days following arterial balloon injury, regional expression of TGF-beta 1 mRNA was assessed using in situ hybridization and the results compared to measures of cellular proliferation and extracellular matrix expansion. Both the TGF-beta 1 concentration measured in culture media of explanted carotid arteries and the quantitative in situ hybridization signal for TGF-beta 1 arterial media and neointima were maximal at 14 days after balloon injury. However, medial cellular proliferation was maximal at 3 days whereas neointimal proliferation was maximal at 14 days and significantly greater than medial proliferation. Neointimal cell density declined significantly between 7 and 14 days, indicating the expansion of extracellular matrix; however, medial cell density was unchanged between 3 and 28 days after balloon injury. Thus, differences in the regional arterial wall relationships between the time course of cellular proliferation, extracellular matrix expansion and the level of TGF-beta 1 expression demonstrate in vivo variability in the response to TGF-beta 1.

All protein kinase C activators are not equivalent. Whereas phorbol 12-myristate 13-acetate (PMA) is the paradigmatic tumor promoter, bryostatin 1 or ingenol 3-angelate are in clinical trials as cancer chemotherapeutic agents. To better understand the structural features contributing to different biological outcomes, we tested a series of phorbol esters differing widely in hydrophobicity in two systems in which bryostatin 1 and PMA give very different responses. With U937 human leukemia cells, PMA inhibits cell growth and induces cell attachment, unlike bryostatin 1. We found that all the phorbol derivatives, like PMA, inhibited growth in a dose dependent manner. They likewise all induced attachment, a measure of differentiation, but several (sapintoxin D, phorbol 12, 13-dibenzoate, phorbol 12, 13-dihexanoate) differed in displaying a biphasic curve for this latter response. In the LNCaP cells, PMA inhibits growth and induces tumor necrosis factor alpha (TNF-alpha) secretion, whereas bryostatin 1 does not. We found that inhibition of growth in response to the phorbol esters ranged from full to partial (31-100 % of the PMA response) and the dose response curves ranged from monophasic to steeply biphasic. Similar divergent behavior was observed for induction of TNF-alpha secretion. For example, the secretion induced by phorbol 12, 13-dibenzoate and phorbol 12, 13-didecanoate was only 22% and 40% of the maximal response, respectively, and the response induced by sapintoxin D was very biphasic: 10 and 30 nM drug induced 56-59%, and 3000 and 10,000 nM induced only 8% and 5% of the maximal response. While none of the compounds induced as little response as did bryostatin 1, the results suggest that the difference was more quantitative in this system than qualitative. We conclude, moreover, that the hydrophobicity of the compounds was not the critical determinant of activity. Unlike the biological response, the translocation pattern of GFP-PKC delta did correlate with hydrophobicity, as the more lipophilic compounds PMA, octylindolactam V and phorbol 12, 13-didecanoate induced translocation to the plasma membrane, followed by translocation to internal membranes, while the more hydrophilic compounds translocated GFP-PKC delta mostly to the internal membranes. We thus conclude that the overall pattern of PKC delta translocation by itself cannot predict the different responses observed. Finally, Nano-pro technology, which can fingerprint the phosphorylation pattern of a protein, is revealing that the different phorbol esters induce divergent phosphorylation patterns of PKC delta, the isoform largely responsible for the phorbol ester induced apoptosis and TNF-alpha secretion in the LNCaP cells, and may provide a powerful tool for viewing the integrated outcome of the numerous elements impinging on this kinase following ligand interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2011-4058

Vav1 is a guanine exchange factor (GEF) for the Rho family of GTPases. It plays a pivotal role in T-cell maturation and development, cytoskeleton organization, and oncogenic transformation. The GEF activity of Vav1 is regulated by several factors, including interaction between its catalytic DH domain and its C1 domain. Its C1 domain shows homology with “typical” C1 domains that are sensitive to the second messenger diacylglycerol (DAG) and phorbol esters (PEs), but it is classified as “atypical” based on its unresponsiveness to these ligands. However, crystallographic analysis has shown that, unlike atypical C1 domains (e.g. Raf1) which possess a distorted structure, the Vav1 C1 retains the geometry of the binding cavity. We hypothesized that residues in the vicinity of the binding pocket might interfere with ligand binding. Sequence alignment with typical C1 domains revealed six unique residues situated along the rim of the putative binding cleft in Vav1 C1: Glu9, Glu10, Pro11, Trp22, Thr24, Tyr26.To probe the role of these residues on DAG/PE sensitivity, we first mutated these sites in the potent PE-sensitive C1b domain of PKCΔ to that of the corresponding sites of Vav1 C1, and analyzed the potency of the mutants for PEs. In vitro binding assays showed that 5 of 6 single-site-mutations (except Trp22) caused significant but limited (10-15 fold) reduction in the binding affinity to phorbol 12,13-dibutyrate (PDBU). Introduction of multiple mutations further decreased the affinity, in a cumulative fashion, leading to no detectable binding in the quintuple mutant. Correspondingly, in vivo confocal microscopy revealed that double and triple GFP-tagged mutants showed much slower and weaker plasma membrane translocation in response to PE than did WT C1bΔ, whereas the quintuple mutation was completely unresponsive. Thus, the ligand-insensitivity of Vav1 C1 reflects the combined effects of Glu9, Glu10, Pro11, Thr24, Tyr26 rather than the effect of a single specific residue. Conversely, introducing “reverse” mutations (corresponding to the residues of PKCΔ C1b) into Vav1 C1 generate binding activity. The quintuple (PKCΔ-like) mutation restored the phorbol-ester-sensitivity of Vav1 C1 to the level of the potent PKCΔ C1b both in vitro and in vivo. In addition, the quintuple mutation conferred PE-sensitivity to the full length Vav1, as revealed by translocation studies. Computer modeling suggests that the presence of these residues confers on Vav1 C1 a hydrophilic surface at the tip of the binding cavity (as opposed to the rather lipophilic surface of PKCΔ C1b), thus impeding interactions with the membrane bilayer and hindering the formation of the ternary binding complex of ligand, receptor (C1) and membrane lipid. We speculate that targeting those unique hydrophilic residues with specific DAG/PE analogs may provide a rationale for selectively manipulating Vav1 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3854. doi:10.1158/1538-7445.AM2011-3854