PINCH Proteins Regulate Cardiac Contractility by Modulating Integrin-Linked Kinase-Protein Kinase B Signaling

Abstract

Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or ␤-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor, modulating expression of stretch-responsive genes such as anf and vegf and thereby enabling the heart to adapt its force of contraction to changing hemodynamic needs. Many of the currently known ILK-mediated functions require additional cofactors and interacting proteins. Increasing focus has therefore been put on the roles of the ternary complex comprising the proteins ILK, ␣-, ␤-, or ␥-parvin, and PINCH, PINCH1 or PINCH2 (5,26,30,34).The ILK-parvin-PINCH (IPP) complex provides physical linkage between integrin transmembrane receptors and the actin cytoskeleton and serves as a signaling mediator that transduces mechanical signals to downstream effectors (32). PINCH and parvins do not have catalytic activity but rather function as adapters for other signaling molecules such as Nck-2 and Ras suppressor 1 (RSU-1), through which PINCH can regulate c-Jun N-terminal kinase (JNK) (7,25).In cardiomyocyte cell cultures, the IPP complex is known to regulate integrin-mediated signaling pathways involved in hypertrophic and apoptotic responses (5). In vivo, a role of the IPP complex and specifically of PINCH proteins in the control of cardiac contractility was demonstrated in a recent study using PINCH-deficient mice. Cardiac muscle-specific double knockout of pinch1 and pinch2, but not single deletion of either gene, resulted in heart failure and early postnatal lethality; however, the underlying molecular pathway was ultimately not elucidated (16).We recently found that zebrafish ILK regulates the cardiac stretch response and contractility via protein kinase B (PKB)-VEGF signaling (2). To investigate if PINCH affects IPP-PKB signaling, we analyzed PINCH1 and PINCH2 function in the zebrafish heart. We show...

A critical role for Ras Suppressor-1 (RSU-1) revealed when PINCH-Integrin-linked Kinase (ILK) binding is disrupted

“…In agreement with data from mammalian cell culture and in zebrafish, we show that loss of PINCH affects ILK protein levels and vice versa, indicating that this is an evolutionarily conserved characteristic of proteins in this adhesion complex (Fukuda et al, 2003;Stanchi et al, 2009;Meder et al, 2011). Contrary to these data, a recent publication has claimed that ILK stabilizes PINCH, but that PINCH does not stabilize ILK, basing this conclusion on qualitative immunofluorescence images, where consistent with other published data ILK localization is preserved in a stck null mutant (Clark et al, 2003;Zervas et al, 2011).…”

“…However, we demonstrate that the reduction in ILK that is associated with stck null mutants is comparable to the reduction in ILK observed in ics mutants, which display no deficits in muscle adhesion or viability. Furthermore, a recent study in zebrafish has shown that overexpresion of ILK in a PINCH morphant does not rescue the defects caused by loss of PINCH (Meder et al, 2011). Taken together, the phenotypes associated with loss of PINCH function cannot be fully attributed to a coordinate loss of ILK.…”

“…A recent publication has also demonstrated mutual stabilization of PINCH and ILK in vivo using antisense morpholinos in zebrafish (Meder et al, 2011). We have tested whether this stabilization phenomenon occurs in Drosophila by examining the levels of PINCH-ILK complex constituents in late stage stck and ilk null embryos.…”

“…Based on the results presented here, disruption of mechanisms that involve growth and survival signaling could also contribute to the lethality observed in ics; PINCH Q38A -Flag rescued animals. ILK and PINCH have both been implicated in survival signaling via AKT, and it will be important to determine the contributions of RSU-1 (Fukuda et al, 2003;Xu et al, 2005;Meder et al, 2011). Many defects observed from disruption of integrin adhesion complexes are attributed to improper ILK-PINCH-Parvin complex formation, function, and downstream signaling (Zhang et al, 2002;Legate et al, 2006;Stanchi et al, 2009;Wickström et al, 2010;Zervas et al, 2011).…”

“…First, the N-terminal LIM domain of PINCH interacts directly with the Nterminal ankyrin repeat domain (ANKR) of ILK Tu et al, 1999). Second, depletion of either ILK or PINCH results in reduction in the levels of the other, indicating a mutual stabilization of these two proteins (Fukuda et al, 2003;Stanchi et al, 2009;Meder et al, 2011). Third, targeted disruption of the interaction between PINCH and ILK in mammalian cell culture experiments by a point mutation in LIM1 of PINCH results in disturbances in cell spreading and survival, as well as reduced stability of both PINCH and ILK (Velyvis et al, 2001;Zhang et al, 2002;Xu et al, 2005).…”