Integrin-linked kinase (ILK) is an ankyrinrepeat containing serine-threonine protein kinase capable of interacting with the cytoplasmic domains of integrin ␤1, ␤2, and ␤3 subunits. Overexpression of ILK in epithelial cells disrupts cell-extracellular matrix as well as cell-cell interactions, suppresses suspension-induced apoptosis (also called Anoikis), and stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of ␤-catenin, where the latter associates with a T cell factor͞ lymphocyte enhancer-binding factor 1 (TCF͞LEF-1) to form an activated transcription factor. We now demonstrate that ILK activity is rapidly, but transiently, stimulated upon attachment of cells to fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore, phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane targetted constitutively active Pi(3)K activates ILK in vivo. We also demonstrate here that ILK is an upstream effector of the Pi(3)K-dependent regulation of both protein kinase B (PKB͞AKT) and glycogen synthase kinase 3 (GSK-3). Specifically, ILK can directly phosphorylate GSK-3 in vitro and when stably, or transiently, overexpressed in cells can inhibit GSK-3 activity, whereas the overexpression of kinasedeficient ILK enhances GSK-3 activity. In addition, kinaseactive ILK can phosphorylate PKB͞AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB͞AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB͞AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB͞AKT, and inhibition of GSK-3.

tumor suppressor ͉ phospholipid phosphatase ͉ cell adhesion ͉ serum T he PTEN͞MMAC͞TEP1 tumor suppressor gene located on chromosome 10q23 (1-3) is mutated at high frequency in a wide variety of human cancers, including glioblastoma, melanoma, and carcinomas of the prostate, breast, endometrium, lung, and head and neck (1, 2, 4, 5-11). Germ-line mutations in the PTEN gene are associated with the development of Cowden's disease and Bannayan-Zonana syndrome (12-15). In addition, the phenotype of PTEN-null mice supports the conclusion that PTEN functions as a tumor suppressor gene (16,17). The homozygous disruption of PTEN results in early embryonic lethality, and heterozygous mice display hyperplastic changes in the prostate, skin, and colon similar to those seen in Cowden's disease. The PTEN gene product shares sequence identity with the protein tyrosine phosphatase family and chicken tensin (18). Recombinant PTEN is capable of dephosphorylating both phosphotyrosine and phosphothreonine, but it also can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], the product of phosphatidylinositol 3-kinase (PI3-kinase) (19,20). Because many of the cancer-related mutations have been mapped to the phosphatase catalytic domain, it has been suggested that the phosphatase activity of PTEN is required for its tumor suppressor function. Overexpression of PTEN can suppress growth in soft agar and tumor formation in nude mice (21,22) and also inhibit focal adhesion kinase (FAK), leading to inhibition of cell adhesion and migration (23). However, targeted disruption of PTEN and PTEN mutations in glioblastoma and prostate carcinoma cells result in the serum-and anchorage-independent activation of protein kinase B (PKB)͞Akt probably because of increased levels of PI(3,4,5)P 3 (16,(24)(25)(26). PKB͞Akt suppresses apoptosis via several possible downstream effectors, including phosphorylation and inactivation of BAD (27-29), inactivation of caspase-9 (30), and repression of the forkhead transcription factor (31). Consistent with this, the disruption of PTEN leads to the suppression of apoptosis (16) but also to accelerated cell cycle progression (25,26). Reconstitution with wild-type (WT) PTEN restores apoptotic sensitivity and induces cell cycle arrest (16,26). These new insights into the tumor-suppressive effects of PTEN recently have been reviewed by Cantley and Neel (32).The activation of PKB͞Akt is regulated in a complex manner via phosphorylation of PKB͞Akt on Thr-308 and Ser-473 (33). Although PDK-1 has been shown to phosphorylate Thr-308, it is not clear whether there is a distinct kinase that exclusively phosphorylates Ser-473. It recently has been proposed that PDK-1 acquires Ser-473 phosphorylation activity in the presence of PRK2 peptide (34). However, as yet, there is no evidence that PDK-1 is the only kinase capable of doing this, and the physiological relevance of the conversion of PDK-1 activity toward Ser-473 phosphorylation by the PRK2 peptide has yet to be demonstrated. On the other hand, the int...

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine ␤-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of ␤-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with ␥-interferon activation site-related motifs within the ␤-lactoglobulin promoter, which we show are required for matrix dependence of ␤-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.Cell-matrix interactions are critical for regulating the phenotype of many cells. In mammary gland, basement membrane is necessary for the prolactin-mediated control of lactation (1-3). However, the mechanism by which extracellular matrix (ECM) 1 influences differentiation in mammary epithelial cells has not been elucidated. We have shown previously that functional ␤ 1 integrins are required for mammary differentiation (4) and that the basement membrane component laminin-1 directs milk protein gene transcription coordinately with prolactin (5).The prolactin pathway is driven through the protein tyrosine kinase Jak2 (6 -10) and one of its substrates, the transcription factor Stat5 (11,12). Stat factors associate with cytokine receptors following ligand binding and subsequently become phosphorylated by receptor-associated Jaks. They then dimerize and translocate to the nucleus where they bind specific DNA sequence motifs, thus contributing to transcriptional activation (13)(14)(15).In this paper we examine whether an element of the prolactin signaling pathway is modulated directly by cell-matrix interactions, thereby mediating the ECM control of transcription. Using primary and first passage cultures of mammary epithelial cells, we demonstrate that the activity of the promoter for the milk protein ␤-lactoglobulin (BLG) is dependent on basement membrane and that Stat5 recognition sites within this promoter are required for transcription. Moreover, we show that the ability of Stat5 to bind its cognate DNA sequence within the BLG promoter requires cell interactions with both basement membrane and prolactin. Thus, matrix and cytokine signals converge on a single pathway.Our data demonstrate, for the first time, that the activity of a Stat transcription factor is a target for regulation by ECM. This establishes a novel signaling...

Integrin-mediated interactions between cells and the extracellular matrix play a fundamental role in the development and function of a variety of tissues by triggering intracellular signals that regulate gene expression. In this study, mouse mammary epithelial cells plated on tissue culture plastic were shown to dramatically up-regulate the steady state levels of mRNA encoding the ␣ 1 , ␣ 2 , ␣ 3 , ␣ 5 , ␣ 6 , ␣ 7 , ␣ v , and ␤ 1 integrin subunits, in contrast to cells cultured on a basement membrane matrix or cells in vivo. This pattern of expression was also observed in a mouse mammary epithelial strain, CID-9 and in other mouse cell lines such as MMTE cells and K1735-M2 melanoma cells. The control of integrin expression was mediated at different levels in different cell types. In K1735-M2 cells, transcription of the ␤ 1 integrin gene was influenced by the substratum, although the levels of integrin protein remained similar. In mammary epithelial cells, the rates of ␤ 1 integrin gene transcription were similar, but mRNA and protein levels were higher in cells cultured on plastic than those on basement membrane. For both cell types, the rate of integrin protein turnover was nearly identical in cells cultured on either substratum. Our results demonstrate that extracellular matrix controls the expression of ␤ 1 integrin subunits and that this regulation is exerted at both transcriptional and post-transcriptional levels.During tissue formation, maintenance, and remodeling, extracellular matrix (ECM) 1 has an invaluable role not only in promoting cell motility and anchorage, but also in inducing cell activation and differentiation. It is becoming increasingly clear that cell-matrix interactions, through specific adhesion receptors, trigger biological responses similar to those transduced by growth factors, hormones, or cytokines (1, 2).We previously showed that interactions between mammary cells and the ECM could regulate the expression of ECM molecules themselves (3). In an in vivo environment, mammary epithelial cells interact with a basement membrane, but on an inadequate substratum in tissue culture, such as on a plastic surface, the cells attempt to recreate their basement membrane by transcribing and translating genes coding for ECM proteins such as fibronectin and laminin (3). We have now asked whether the type of cell-matrix interactions also induce a similar regulation in the expression of cell surface ␤ 1 integrin receptors for ECM components. Any matrix-induced changes in the levels and patterns of integrin subunits might alter the way that microenvironmental signals are perceived, trigger the expression of new sets of genes, and modify cell phenotype.We therefore examined the mRNAs coding for different subunits of the ␤ 1 integrin family in mammary epithelial cells and compared their expression levels in cells cultured either on a plastic surface or on a laminin-enriched reconstituted basement membrane matrix. We then assessed whether changes in the amounts of mRNA were reflected at the protein level....

We have shown previously that switching of the alpha v-associated beta1 and beta5 integrin subunits during differentiation of myelin-forming oligodendrocytes may regulate important aspects of cell behaviour such as migration (Milner et al., 1996: J Neurosci 16:7240-7252). In this study we have examined the developmental regulation of other alpha v-associated beta subunits in oligodendroglial cell cultures and also the control of their expression by neurons, using xenocultures to distinguish glial and neuronal integrins. We have found that oligodendroglia express alpha vbeta8 in addition to the previously-described alpha vbeta1, alpha vbeta3, and alpha vbeta5. Beta8 and beta3 together comprise the 80kD band seen in alpha v immunoprecipitations that represents the most abundant alpha v-associated beta subunit and show reciprocal patterns of expression during development. Alpha vbeta8 is expressed at high levels on oligodendrocyte precursors and differentiated oligodendrocytes but diminishes during the intermediate stages of differentiation. Alpha vbeta3, in contrast, shows an opposite pattern of expression, with the highest levels seen at the intermediate stages of differentiation and little expression on either oligodendrocyte precursors and differentiated oligodendrocytes. The expression of alpha vbeta3 is not altered by coculture with neurons, unlike that of alpha vbeta8, in which the decrease seen at the intermediate stages of differentiation is less marked in the presence of neurones. Our results confirm that switching of alpha v-associated beta subunits is an important feature of oligodendrocyte differentiation and suggest that alpha vbeta8 and alpha vbeta3 have distinct functions during myelination.

CD44 and sulfation have both been implicated in leukocyte adhesion. In monocytes, the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) stimulates CD44 sulfation, and this correlates with the induction of CD44-mediated adhesion events. However, little is known about the sulfation of CD44 or its induction by inflammatory cytokines. We determined that TNF-alpha induces the carbohydrate sulfation of CD44. CD44 was established as a major sulfated cell surface protein on myeloid cells. In the SR91 myeloid cell line, the majority of CD44 sulfation was attributed to the glycosaminoglycan chondroitin sulfate. However, TNF-alpha stimulation increased CD44 sulfation two- to threefold, largely attributed to the increased sulfation of N- and O-linked glycans on CD44. Therefore, TNF-alpha induced a decrease in the percentage of CD44 sulfation due to chondroitin sulfate and an increase due to N- and O-linked sulfation. Furthermore, TNF-alpha induced the expression of 6-sulfo N-acetyl lactosamine (LacNAc)/Lewis x on these cells, which was detected by a monoclonal antibody after neuraminidase treatment. This 6-sulfo LacNAc/Lewis x epitope was induced on N-linked and (to a lesser extent) on O-linked glycans present on CD44. This demonstrates that CD44 is modified by sulfated carbohydrates in myeloid cells and that TNF-alpha modifies both the type and amount of carbohydrate sulfation occurring on CD44. In addition, it demonstrates that TNF-alpha can induce the expression of 6-sulfo N-acetyl glucosamine on both N- and O-linked glycans of CD44 in myeloid cells.

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1b and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1b on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.

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