Integrin Dependence of Brain Natriuretic Peptide Gene Promoter Activation by Mechanical Strain

Faquan Liang, Amha Atakilit, David G. Gardner
2000 Journal of Biological Chemistry  
Expression of the brain natriuretic peptide (BNP) gene in cultured neonatal rat ventricular myocytes is activated by mechanical strain in vitro. We explored the role of cell-matrix contacts in initiating the strain-dependent increment in human BNP (hBNP) promoter activity. Coating the culture surface with fibronectin effected a dose-dependent increase in basal hBNP luciferase activity and amplification of the response to strain. Preincubation of myocytes with an RGD peptide (GRGDSP) or with
more » ... GRGDSP) or with soluble fibronectin, each of which would be predicted to compete for cell-matrix interactions, resulted in a dose-dependent reduction in straindependent hBNP promoter activity. A functionally inert RGE peptide (GRGESP) was without effect. Using fluorescence-activated cell sorting, we demonstrated the presence of ␤ 1 , ␤ 3 , and ␣ v ␤ 5 integrins in myocytes as well as non-myocytes and ␣1 only in non-myocytes in our cultures. Inclusion of antibodies directed against ␤ 1 , ␤ 3 , or ␣ v ␤ 5 , but not ␣ 1 , ␣ 2 , or cadherin, was effective in blocking the BNP promoter response to mechanical strain. These same antibodies (anti-␤ 3 , -␤ 1 , and -␣ v ␤ 5 ) had a similar inhibitory effect on strain-stimulated ERK, p38 MAPK, and, to a lesser extent, JNK activities in these cells. Cotransfection with chimeric integrin receptors capable of acting as dominant-negative inhibitors of integrin function demonstrated suppression of straindependent BNP promoter activity when vectors encoding ␤ 1 or ␤ 3 , but not ␤ 5 , ␣ 5 , or a carboxyl-terminal deletion mutant of ␤ 3 (␤ 3 B), were employed. These studies underscore the importance of cell-matrix interactions in controlling cardiac gene expression and suggest a potentially important role for these interactions in signaling responses to mechanical stimuli within the myocardium. Brain natriuretic peptide (BNP) 1 is a vasoactive hormone that, despite its name, is produced primarily in the heart. Like atrial natriuretic peptide (ANP), it possesses potent natriuretic, diuretic, and vasorelaxant activities, properties that position it physiologically as a potential antagonist of vasoactive systems (e.g. the renin-angiotensin and sympathetic nervous systems) associated with intravascular volume expansion and increased blood pressure (1). Like ANP, BNP is expressed in both atrial and ventricular myocardia, although differential expression is not so marked as that for ANP (the atrial/ventricular ratio for BNP expression is ϳ3:1, whereas that for ANP is in the range of 40:1) (2). As with ANP, ventricular expression of BNP is activated in pathophysiological states associated with hemodynamic overload in vivo (3, 4) and following exposure to hypertrophy-promoting maneuvers in vitro (5-10). In the latter group are a number of biochemical (e.g. ␣-adrenergic agonists, endothelin, angiotensin II, and cardiotrophin-1) as well as physical (e.g. mechanical strain and hypoxia) stimuli that trigger increases in cell size and protein synthesis, reactivate a program of fetal gene expression, and reorganize sarcomeric structure of myocytes in culture. These phenotypic changes closely resemble those associated with myocyte hypertrophy in vivo (11). A number of recent studies have documented the importance of the extracellular matrix in establishing cellular responses, both qualitatively and quantitatively, to a variety of biochemical and physical stimuli (12, 13). In the case of mechanical strain, it has been hypothesized that key matrix-integrin attachments may participate in the signal transduction process linking the strain signal to changes in gene expression, cytoskeletal reorganization, and DNA synthesis (14) . We have recently shown that application of cyclical, passive mechanical strain (i.e. stretch) to cultured neonatal rat ventricular myocytes in vitro results in stimulation of immunoreactive BNP secretion, increased steady-state levels of the BNP gene transcript, and activation of a transfected human BNP (hBNP) gene promoter (8). In the present study, we show that both basal and strain-activated BNP promoter activities are dependent upon specific contacts that the ventricular myocyte makes with proteins in the extracellular matrix, implying a potential signaling function for these contacts in the subsequent activation of gene expression. (15) . Purified hamster anti-rat/mouse ␣ 1 , ␣ 2 , ␣ 5 , and ␤ 1 monoclonal antibodies; purified mouse anti-rat ␤ 3 monoclonal antibody; FITC-labeled anti-hamster IgG; and FITC-conjugated hamster anti-rat ␤ 1 monoclonal antibody were obtained from Pharmingen (San Diego,
doi:10.1074/jbc.m001660200 pmid:10764770 fatcat:4avky2tc2nd7hncfypipxrc6za