Signaling mechanisms underlying strain-dependent brain natriuretic peptide gene transcription.

Activation of brain natriuretic peptide (BNP) gene promoter activity represents one of the earliest and most reliable markers of ventricular cardiac myocyte hypertrophy. We recently demonstrated that mechanical strain increases immunoreactive BNP secretion, steady-state BNP mRNA levels and BNP gene transcriptional activity in neonatal rat myocyte cultures. We have also shown that strain-dependent BNP gene transcription is critically dependent on the functional integrity of a number of integrins (specfically beta1, beta3, and alpha(v)beta5 integrins) present on the surface of cardiac myocytes. When used alone, each of these antibodies resulted in a significant reduction in strain-dependent activation of a transfected hBNP-luciferase reporter and inhibition of a number of signaling pathways that have been linked to stimulation of this reporter (e.g., extracellular signal regulated kinase and c-Jun amino terminal kinase). The present study shows that combinations of these antibodies resulted in further reductions in hBNP gene promoter activity and inhibition of the relevant signaling cascades. These studies provide further support for the importance of integrin-matrix interactions in promoting strain-dependent changes in cardiac myocyte gene transcription.

Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac myocyte hypertrophy.

Hypertrophic terminally differentiated cardiac myocytes show increased sarcomeric organization and altered gene expression. Previously, we established a role for the nonreceptor tyrosine kinase Src in signaling cardiac myocyte hypertrophy. Here we report evidence that p130Cas (Cas) and focal adhesion kinase (FAK) regulate this process. In neonatal cardiac myocytes, tyrosine phosphorylation of Cas and FAK increased upon endothelin (ET) stimulation. FAK, Cas, and paxillin were localized in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. Cas, alone or in cooperation with Src, modulated basal and ET-stimulated atrial natriuretic peptide (ANP) gene promoter activity, a marker of cardiac hypertrophy. Expression of the C-terminal focal adhesion-targeting domain of FAK interfered with localization of endogenous FAK to Z-lines. Expression of the Cas-binding proline-rich region 1 of FAK hindered association of Cas with FAK and impaired the structural stability of sarcomeres. Collectively, these results suggest that interaction of Cas with FAK, together with their localization to Z-lines, is critical to assembly of sarcomeric units in cardiac myocytes in culture. Moreover, expression of the focal adhesion-targeting and/or the Cas-binding proline-rich regions of FAK inhibited ANP promoter activity and suppressed ET-induced ANP and brain natriuretic peptide gene expression. In summary, assembly of signaling complexes that include the focal adhesion proteins Cas, FAK, and paxillin at Z-lines in the cardiac myocyte may regulate, either directly or indirectly, both cytoskeletal organization and gene expression associated with cardiac myocyte hypertrophy.

The lipid peroxidation product, 4-hydroxy-2-trans-nonenal, alters the conformation of cortical synaptosomal membrane proteins.

Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta). A beta-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2-trans-nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of A beta and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1-10 microM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and A beta-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 microM). The results obtained are discussed with relevance to the hypothesis of A beta-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.