Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3␤ on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation. PKB/Akt1 is activated in a PI 3-kinase-dependent manner and regulates cell survival, proliferation, differentiation, motility, and metabolism (1). The activity of PKB/Akt is constitutively activated under situations of chronic activation of PI 3-kinase, for example, by the mutational inactivation of the tumor suppressor PTEN. PKB/Akt regulates apoptosis and cell cycle progression by promoting the phosphorylation of proapoptotic proteins such as Bad, Forkhead transcription factors, and the cell cycle inhibitor p27(kip1) (2-5).The full activation of PKB/Akt requires phosphorylation on Thr-308 and Ser-473 (6). Whereas PDK-1 has been demonstrated to phosphorylate PKB/Akt on Thr-308 (7), the kinase responsible for phosphorylation at the hydrophobic Ser-473 site (PDK-2) has not been identified despite extensive efforts. PDK-1 was initially proposed as also being responsible for the stimulation of phosphorylation of Ser-473 (8). However, this site is inducibly phosphorylated in PDK-1 knock-out cells, pointing to the existence of a distinct Ser-473 kinase (9). Autophosphorylation is another proposed mechanism for the phosphorylation on Ser-473 (10). The integrin-linked kinase (ILK), an integrin-and growth factor-regulated PI 3-kinase-dependent kinase (11,12), has also been shown to promote the phosphorylation of PKB/Akt on Ser-473 but not on [11][12][13][14][15]. Growth factor-and extracellular matrix-induced PKB/ Akt Ser-473 phosphorylation is inhibited by kinase-deficient, dominant-negative ILK (1,13,14), as well as by small molecule ILK kinase inhibitors (14, 16). Inhibition of ILK activ...

A requirement for integrin-mediated adhesion in cardiac physiology is revealed through targeted deletion of integrin-associated genes in the murine heart. Here we show that targeted ablation of the integrin-linked kinase (ILK) expression results in spontaneous cardiomyopathy and heart failure by 6 wk of age. Deletion of ILK results in disaggregation of cardiomyocytes, associated with disruption of adhesion signaling through the ␤1-integrin/FAK (focal adhesion kinase) complex. Importantly, the loss of ILK is accompanied by a reduction in cardiac Akt phosphorylation, which normally provides a protective response against stress. Together, these results suggest that ILK plays a central role in protecting the mammalian heart against cardiomyopathy and failure.Supplemental material is available at http://www.genesdev.org.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25-dihydroxyvitaminD3 (1,25(OH)2D3). Mice in which the 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene was inactivated presented the same clinical phenotype as patients with PDDR and were used to study renal expression of the epithelial Ca2+ channel (ECaC1), the calbindins, Na+/Ca2+ exchanger (NCX1), and Ca2+-ATPase (PMCA1b). Serum Ca2+ (1.20+/-0.05 mM) and mRNA/protein expression of ECaC1 (41+/-3%), calbindin-D28K (31+/-2%), calbindin-D9K (58+/-7%), NCX1 (10+/-2%), PMCA1b (96+/-4%) were decreased in 1alpha-OHase-/- mice compared with 1alpha-OHase+/- littermates. Feeding these mice a Ca2+-enriched diet normalized serum Ca2+ levels and expression of Ca2+ proteins except for calbindin-D9K expression. 1,25(OH)2D3 repletion resulted in increased expression of Ca2+ transport proteins and normalization of serum Ca2+ levels. Localization of Ca2+ transport proteins was clearly polarized in which ECaC1 was localized along the apical membrane, calbindin-D28K in the cytoplasm, and calbindin-D9K along the apical and basolateral membranes, resulting in a comprehensive mechanism facilitating renal transcellular Ca2+ transport. This study demonstrated that high dietary Ca2+ intake is an important regulator of the renal Ca2+ transport proteins in 1,25(OH)2D3-deficient status and thus contributes to the normalization of blood Ca2+ levels.

Integrin-linked kinase (ILK) has been implicated in the pathogenesis of proteinuria and congenital nephrotic syndrome. However, the function of ILK in glomerular podocyte in a physiologic setting remains unknown. In this study, a mouse model was generated in which ILK gene was selectively disrupted in podocytes by using the Cre-LoxP system. Podocyte-specific ablation of ILK resulted in heavy albuminuria, glomerulosclerosis, and kidney failure, which led to animal death beginning at 10 wk of age. Podocyte detachment and apoptosis were not observed at 4 wk of age, when albuminuria became prominent, indicating that they are not the initial cause of proteinuria. Electron microscopy revealed an early foot process effacement, as well as morphologic abnormality, in ILK-deficient podocytes. ILK deficiency caused an aberrant distribution of nephrin and ␣-actinin-4 in podocytes, whereas the localization of podocin and synaptopodin remained relatively intact. Co-immunoprecipitation demonstrated that ILK physically interacted with nephrin to form a ternary complex, and ␣-actinin-4 participated in ILK/nephrin complex formation. Therefore, ILK plays an essential role in specifying nephrin and ␣-actinin-4 distribution and in maintaining the slit diaphragm integrity and podocyte architecture. These results also illustrate that the integrin and slit diaphragm signals in podocytes are intrinsically coupled through an ILK-dependent mechanism.

Abstract. Estrogen deficiency results in a negative Ca 2ϩ balance and bone loss in postmenopausal women. In addition to bone, the intestine and kidney are potential sites for estrogen action and are involved in Ca 2ϩ handling and regulation. The epithelial Ca 2ϩ channel ECaC1 (or TRPV5) is the entry channel involved in active Ca 2ϩ transport. Ca 2ϩ entry is followed by cytosolic diffusion, facilitated by calbindin-D 28K and/or calbindin-D 9k , and active extrusion across the basolateral membrane by the Na ϩ /Ca 2ϩ -exchanger (NCX1) and plasma membrane Ca 2ϩ -ATPase (PMCA1b). In this transcellular Ca 2ϩ transport, ECaC1 probably represents the final regulatory target for hormonal control. The aim of this study was to determine whether 17␤-estradiol (17␤-E 2 ) is involved in Ca 2ϩ reabsorption via regulation of the expression of ECaC1. The ovariectomized rat model was used to investigate the regulation of ECaC1, at the mRNA and protein levels, by 17␤-E 2 replacement therapy. Using real-time quantitative PCR and immunohistochemical analyses, this study demonstrated that 17␤-E 2 treatment at pharmacologic doses increased renal mRNA levels of ECaC1, calbindin-D 28K , NCX1, and PMCA1b and increased the protein abundance of ECaC1. Furthermore, the involvement of 1,25-dihydroxyvitamin D 3 in the effects of 17␤-E 2 was examined in 25-hydroxyvitamin D 3 -1␣-hydroxylase-knockout mice. Renal mRNA expression of calbindin-D 9K , calbindin-D 28K , NCX1, and PMCA1b was not significantly altered after 17␤-E 2 treatment. In contrast, ECaC1 mRNA and protein levels were both significantly upregulated. Moreover, 17␤-E 2 treatment partially restored serum Ca 2ϩ levels, from 1.63 Ϯ 0.06 to 2.03 Ϯ 0.12 mM. In conclusion, this study suggests that 17␤-E 2 is positively involved in renal Ca 2ϩ reabsorption via the upregulation of ECaC1, an effect independent of 1,25-dihydroxyvitamin D 3 .