Roy Zent

THE TALIN HEAD DOMAIN BINDS TO INTEGRIN BETA SUBUNIT CYTOPLASMIC TAILS AND REGULATES INTEGRIN ACTIVATION

The β subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to β integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of β1A, β1D and β3integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin β tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin αIIbβ3; activation was dependent on the presence of both the talin head domain and the integrin β3 cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

The tail of integrins, talin, and kindlins.

Tales of Talin, Kindlin, and Integrin The integrins are receptors on the surface of animal cells that mediate attachment to the extracellular matrix. Integrins also act as signaling molecules, activating signaling pathways when they bind to their ligands in the matrix. Furthermore, integrins can communicate signals from the inside to the outside of the cell when signals within the cell alter the affinity of integrins for their extracellular ligands. Moser et al. (p. 895) review recent advances in understanding the roles of the proteins talin and kindlin in such bidirectional signaling and how they influence the function of integrins in health and disease. Integrins are transmembrane cell–adhesion molecules that carry signals from the outside to the inside of the cell and vice versa. Like other cell surface receptors, integrins signal in response to ligand binding; however, events within the cell can also regulate the affinity of integrins for ligands. This feature is important in physiological situations such as those in blood, in which cells are always in close proximity to their ligands, yet cell-ligand interactions occur only after integrin activation in response to specific external cues. This review focuses on the mechanisms whereby two key proteins, talin and the kindlins, regulate integrin activation by binding the tails of integrin-β subunits.

Kindlin-2 controls bidirectional signaling of integrins.

Control of integrin activation is required for cell adhesion and ligand-induced signaling. Here we report that loss of the focal adhesion protein Kindlin-2 in mice results in peri-implantation lethality caused by severe detachment of the endoderm and epiblast from the basement membrane. We found that Kindlin-2-deficient cells were unable to activate their integrins and that Kindlin-2 is required for talin-induced integrin activation. Furthermore, we demonstrate that Kindlin-2 is required for integrin outside-in signaling to enable firm adhesion and spreading. Our findings provide evidence that Kindlin-2 is a novel and essential element of bidirectional integrin signaling.

Sirt1 activation protects the mouse renal medulla from oxidative injury

Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. Here, we set out to investigate the role of Sirt1 in the kidney. Our initial analysis indicated that it was abundantly expressed in mouse renal medullary interstitial cells in vivo. Knocking down Sirt1 expression in primary mouse renal medullary interstitial cells substantially reduced cellular resistance to oxidative stress, while pharmacologic Sirt1 activation using either resveratrol or SRT2183 improved cell survival in response to oxidative stress. The unilateral ureteral obstruction (UUO) model of kidney injury induced markedly more renal apoptosis and fibrosis in Sirt1+/- mice than in wild-type controls, while pharmacologic Sirt1 activation substantially attenuated apoptosis and fibrosis in wild-type mice. Moreover, Sirt1 deficiency attenuated oxidative stress-induced COX2 expression in cultured mouse renal medullary interstitial cells, and Sirt1+/- mice displayed reduced UUO-induced COX2 expression in vivo. Conversely, Sirt1 activation increased renal medullary interstitial cell COX2 expression both in vitro and in vivo. Furthermore, exogenous PGE2 markedly reduced apoptosis in Sirt1-deficient renal medullary interstitial cells following oxidative stress. Taken together, these results identify Sirt1 as an important protective factor for mouse renal medullary interstitial cells following oxidative stress and suggest that the protective function of Sirt1 is partly attributable to its regulation of COX2 induction. We therefore suggest that Sirt1 provides a potential therapeutic target to minimize renal medullary cell damage following oxidative stress.

Activated type I TGFβ receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression

We have examined the effects of transforming growth factor-beta (TGFβ) signaling on mammary epithelial cell survival. Transgenic mice expressing an active mutant of Alk5 in the mammary gland (MMTV-Alk5T204D) exhibited reduced apoptosis in terminal endbuds and during postlactational involution. Transgene-expressing mammary cells contained lower Smad2/3 and higher c-myc levels than controls, high ligand-independent phosphatidylinositol-3 kinase (PI3K) and Akt activities, and were insensitive to TGFβ-mediated growth arrest. Treatment with a proteasome inhibitor increased Smad2/3 levels and ligand-independent Smad transcriptional reporter activity, as well as reduced both c-myc protein and basal cell proliferation. Treatment with an Alk5 kinase small-molecule inhibitor upregulated Smad2/3 levels, reduced PI3K activity, P-Akt, and c-myc, and inhibited cell survival. Although Alk5T204D-expressing mice did not develop mammary tumors, bigenic MMTV-AlkT204D × Neu mice developed cancers that were more metastatic than those occurring in MMTV-Neu transgenics. These data suggest that (1) TGFβ can signal to PI3K/Akt and enhance mammary epithelial cell survival in vivo before cytological or histological evidence of transformation, and (2) TGFβ signaling can provide epithelial cells with a ‘gain-of-function’ effect that synergizes with oncogene-induced transformation.

Integrin-linked kinase is an adaptor with essential functions during mouse development

The development of multicellular organisms requires integrin-mediated interactions between cells and their extracellular environment. Integrin binding to extracellular matrix catalyses assembly of multiprotein complexes, which transduce mechanical and chemical signals that regulate many aspects of cell physiology. Integrin-linked kinase (Ilk) is a multifunctional protein that binds β-integrin cytoplasmic domains and regulates actin dynamics by recruiting actin binding regulatory proteins such as α- and β-parvin. Ilk has also been shown to possess serine/threonine kinase activity and to phosphorylate signalling proteins such as Akt1 and glycogen synthase kinase 3β (Gsk3β) in mammalian cells; however, these functions have been shown by genetic studies not to occur in flies and worms. Here we show that mice carrying point mutations in the proposed autophosphorylation site of the putative kinase domain and in the pleckstrin homology domain are normal. In contrast, mice with point mutations in the conserved lysine residue of the potential ATP-binding site of the kinase domain, which mediates Ilk binding to α-parvin, die owing to renal agenesis. Similar renal defects occur in α-parvin-null mice. Thus, we provide genetic evidence that the kinase activity of Ilk is dispensable for mammalian development; however, an interaction between Ilk and α-parvin is critical for kidney development.

Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel

Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10-/- mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10-/- mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease.

β1 integrin expression by podocytes is required to maintain glomerular structural integrity

Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus.

Prostaglandin E2-EP4 Receptor Promotes Endothelial Cell Migration via ERK Activation and Angiogenesis in Vivo

Prostaglandin E2 (PGE2), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1–4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4flox/flox mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE2 or the EP4 selective agonists PGE1-OH and ONO-AE1–329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4flox/flox cells, no effects were seen in adenoCre-transduced EP4flox/flox cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE2-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE2, as well as PGE1-OH and ONO-AE1–329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.

Distinct Domains of CD98hc Regulate Integrins and Amino Acid Transport

CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin β1A cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hcs effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein.