Dietmar von der Ahe

Molecular Characterization of Human Zyxin

Zyxin is a component of adhesion plaques that has been suggested to perform regulatory functions at these specialized regions of the plasma membrane. Here we describe the isolation and characterization of cDNAs encoding human and mouse zyxin. Both the human and mouse zyxin proteins display a collection of proline-rich sequences as well as three copies of the LIM domain, a zinc finger domain found in many signaling molecules. The human zyxin protein is closely related in sequence to proteins implicated in benign tumorigenesis and steroid receptor binding. Antibodies raised against human zyxin recognize an 84-kDa protein by Western immunoblot analysis. The protein is localized at focal contacts in adherent erythroleukemia cells. By Northern analysis, we show that zyxin is widely expressed in human tissues. The zyxin gene maps to human chromosome 7q32-q36.

MEK hyperphosphorylation coincides with cell cycle shut down of cultured smooth muscle cells

Smooth muscle cells (SMCs) form the backbone of arteries and their proliferation hallmarks collateral vessel growth, a process termed arteriogenesis, as well as pathogenic responses such as restenosis. Since signaling pathways in SMCs are the main targets for therapeutic interventions, we aimed to determine how and to what extent the activation of the ubiquitous MEK–ERK signaling pathway correlates with important in vivo phenomena such as dedifferentiation, nuclear activation and proliferation of SMCs. Specificity of this pathway was monitored using MEK inhibitors UO126 and PD98059 in platelet derived growth factor‐AB (PDGF‐AB)‐ and fibroblast growth factor‐2 (FGF‐2)‐stimulated SMCs. PDGF‐AB induced a rapid MEK activation followed by phosphorylation of the MEK substrates ERK1/2 while FGF‐2 showed a less pronounced and delayed activation. Both growth factors triggered a marked phosphorylation of c‐Myc and expression of Egr1. Pretreatment with MEK inhibitors suppressed the activation of the ERK cascade, abolished the down‐regulation of desmin and led to cell cycle arrest. However, the reversibility of p27Kip1 down‐regulation by UO126 was mainly observed after PDGF‐AB stimulation, indicating MEK independent p27Kip1 down‐regulation by FGF‐2. Surprisingly, treatment of SMCs with UO126 or PD98059 increased the level of MEK phosphorylation in a dose dependent manner at serine residues 217/221 in the presence as well as in the absence of both growth factors. Our results strongly imply that depending on the environmental context phosphorylation of serines 217/221 serves as an “on” as well as an “off ” switch.

Porcine aortic endothelial cells show little effects on smooth muscle cells but are potent stimulators of cardiomyocyte growth

Smooth muscle cells (SMC) and endothelial cells (EC) play a pivotal role in arteriogenesis and atherosclerosis. We evaluated the role of EC on the growth of SMC and neonatal cardiomyocytes (NEO) by using serum-free EC-supernatant (AoCM). Five percent fetal calf serum was used in order to mimic growth effects of blood. EC and SMC purities were 99% as determined by absence or presence of markers such as CD31, desmin, α-smooth muscle actin and tropomyosin using immunostaining and FACS analysis. AoCM markedly influenced the morphology of NEO as determined by α-actinin staining but showed only little effect on the phenotype of SMC. Protein synthesis after 2 days increased 2.5-fold in SMC and 3.7-fold in NEO as determined by tritium incorporation. The values for serum (2.8 and 2.3-fold, respectively) were comparable. The induction of DNA-synthesis by serum in NEO was twice that of AoCM (3.9-fold). However, proliferative effects of serum and AoCM on SMC differed markedly: Serum induced a 66-fold increase in DNA-synthesis resulting in a 54% higher cell number. DNA-synthesis after AoCM treatment lead to a nonsignificant small increase and no proliferation was detected. Platelet derived growth factor (PDGF-AB), present in blood, induced a 47-fold increase in DNA-synthesis and a 38% increase in cell number. Our data suggest that EC in the absence of physical forces exert strong morphogenic effects on cardiomyocytes but they lack specific effects on smooth muscle cells. In vessels EC might function as a border to isolate SMC from key regulators in blood such as PDGFs.