We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1␤ (IL-1␤), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1␤-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycinearginine-glycine-aspartic acid-serine)-pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat ␤ 3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1␤ increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1␤-stimulated increases in translocation of PKC-from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-using PKCpseudosubstrate inhibited IL-1␤-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via ␤ 3 integrins, inhibits IL-1␤-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.The dynamic synthesis and breakdown of extracellular matrix (ECM) 1 proteins may play an important role in myocardial remodeling (1, 2). Osteopontin (OPN) is a glycosylated and phosphorylated ECM protein (3, 4). Although first isolated from mineralized matrix, OPN has since been shown to be synthesized by cardiac fibroblasts, endothelial cells, and myocytes (3, 5-7). Using spontaneously hypertensive and aorticbanded rats, we have shown increased expression of OPN coincident with heart failure (8). OPN interacts with specific integrins, namely, and also with CD44 receptors and affects many cellular processes including cell attachment, spreading, and migration (3, 4, 9). Recently, we have documented an increased expression of OPN in remote and infarct regions of left ventricle following myocardial infarction (MI). Furthermore, using OPN knock-out mice and MI as a model of myocardial remodeling, we demonstrated that a lack of OPN results in greater left ventricle dilation and reduced collagen synthesis and accumulation 1 month post-MI, suggesting a role for OPN in ECM reorganization in the heart during myocardial remodeling (10). A critical role for OPN in the generation of interstitial fibrosis was observed in the skin incision model of wound healing and in the kidney following obstructive...

No abstract

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with β1 or β3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

No abstract