Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility.

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin-/-) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal-regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin-FAK interactions. A vinculin fragment (amino acids 811-1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin-/- cells. Both vinY822F and vin-/- cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin-/- and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.

Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells.

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.

Early growth response 1 acts as a tumor suppressor in vivo and in vitro via regulation of p53.

The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFbeta1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop.

The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Co-factors p300 and CBP catch Egr1 in their network.

p300 and CBP are large (300 kDa) nuclear scaffold proteins that act as co-activators of transcription in most cell types and are over-expressed in prostate cancer. Recently, the Egr1 transcription factor was shown to up- or down-regulate p300 and CBP transcription based on the nature of its post-translational modification. Notably, interactions of the three proteins provide fine tuning for Egr1-induced growth or cell death responses.

Vinculin controls PTEN protein level by maintaining the interaction of the adherens junction protein beta-catenin with the scaffolding protein MAGI-2.

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role.

Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival.

The transcription factor, Egr1, so-called because it is encoded by the immediate early growth response gene, Egr1, is rapidly induced by growth factors to transduce the proliferative signal. The induction of Egr1 by external stimuli is generally transient but appears to be sustained in some prostate tumor cell lines and tumors, suggesting that Egr1 stimulates tumor cell growth. In contradiction, in breast, lung and brain tumors, Egr1 expression is often absent or reduced and when re-expressed, results in growth suppression. Re-expression of Egr1 in tumor cells also leads to antiapoptotic activity, which would encourage tumor cell survival. Egr1 is also required for, or stimulates, the differentiation of several cell types. Another contradiction is that after stress stimuli to some cell types, Egr1 is required for programmed cell death or apoptosis in both normal and tumor cells. Egr1 also plays a role in tumor progression, through the hypoxic signal generated in growing tumors. Egr1 is highly induced under these conditions and its activities stimulate angiogenesis and improved survival of tumor cells. How this large agenda can be achieved lies in the choice of Egr1 target genes, and varying patterns of coordinated expression have been described, but the mechanisms for this choice are not clear. This review points to areas where research should be focussed.

Cripto: a tumor growth factor and more.

Cripto, a growth factor with an EGF-like domain, and the first member of the EGF-CFC family of genes to be sequenced and characterized, contributes to deregulated growth of cancer cells. A role for Cripto in tumor development has been described in the human and the mouse. Members of the EGF-CFC family are found only in vertebrates: CFC proteins in zebrafish, Xenopus, chick, mouse and human have been characterized and indicate some common general functions in development. Cripto expression was first found in human and mouse embryonal carcinoma cells and male teratocarcinomas, and was demonstrated to be over-expressed in breast, cervical, ovarian, gastric, lung, colon, and pancreatic carcinomas in contrast to normal tissues where Cripto expression was invariably low or absent. Cripto may play a role in mammary tumorigenesis, since in vitro, Cripto induces mammary cell proliferation, reduces apoptosis, increases cell migration, and inhibits milk protein expression. This prediction is strengthened by observations of Cripto expression in 80% of human and mouse mammary tumors. At least three important roles for Cripto in development have created considerable interest, and each activity may be distinct in its mechanism of receptor signaling. One role is in the patterning of the anterior-posterior axis of the early embryo, a second is a crucial role in the development of the heart, and a third is in potentiating branching morphogenesis and modulating differentiation in the developing mammary gland. Whether these properties are functions of different forms of Cripto, different Cripto receptors or the distinct domains within this 15-38 kDa glycoprotein are examined here, but much remains to be revealed about this evolutionarily conserved gene product. Since all Cripto receptors have not yet been determined with certainty, future possible uses as therapeutic targets remain to be developed. Cripto is released or shed from expressing cells and may serve as an accessible marker gene in the early to mid-progressive stages of breast and other cancers. Meanwhile some speculations on possible receptor complexes for Cripto signaling in mammary cells are offered here as a spur to further discoveries.

In vivo cloning and characterization of a new growth suppressor protein TOE1 as a direct target gene of Egr1.

Egr1, an immediate early transcription factor, responds to diverse stimuli and affects gene transcription to accomplish its biological effects. One important effect of Egr1 expression is to decrease the growth and tumorigenic potential of several tumor cell types. To identify important Egr1 target genes, we have adapted a methodology involving formaldehyde-induced protein-DNA cross-linking, chromatin immunoprecipitation, and multiplex PCR. Using this approach, we report the cloning of a new Egr1 target gene that is able to account, at least in part, for the growth inhibitory activity of Egr1. We have named this new protein TOE1 for target of Egr1.

Coactivating factors p300 and CBP are transcriptionally crossregulated by Egr1 in prostate cells, leading to divergent responses.

Related coactivators p300 and CBP affect the transcriptional activities of many transcription factors (TF), producing multiple downstream effects. Here we show that immediate early response TF, Egr1, acts upstream of p300/CBP to induce or to repress transcription, depending on the stimulus. Cells induced with serum to increase endogenous Egr1 increase the transcription of p300/CBP only when Egr1 binding sites in the promoter are not mutated, causing the expression of downstream targets of Egr1 which leads to survival and growth. Induction of p300/CBP by Egr1 results in acetylation and stabilization of Egr1 and transactivation of survival genes but repression of Egr1 and p300/CBP in negative feedback loops. In contrast, induction of Egr1 by UV-C irradiation leads to repression of p300/CBP transcription: Egr1 is preferentially phosphorylated, leading to regulation of target genes that cause cell death. This complex balance of opposing effects appears to finely modulate important cellular life and death responses.

Egr1 promotes growth and survival of prostate cancer cells. Identification of novel Egr1 target genes.

In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells.

Heterozygous inactivation of the vinculin gene predisposes to stress-induced cardiomyopathy.

Vinculin and its muscle splice variant metavinculin link focal adhesions and cell-to-cell contact sites to the actin cytoskeleton. We hypothesized that normal expression of vinculin isoforms would be essential for integrity of cardiomyocytes and preservation of normal cardiac function. We studied heterozygous vinculin knockout mice (Vin+/-) that develop and breed normally. The Vin+/- mice displayed: 1) a 58% reduction of vinculin and a 63% reduction of metavinculin protein levels versus wild-type littermates; 2) normal basal cardiac function and histology but abnormal electrocardiograms, intercalated disks, and ICD-related protein distribution; 3) increased mortality following acute hemodynamic stress imposed by transverse aortic constriction (TAC); 4) cardiac dysfunction by 6 weeks post-TAC; and 5) misalignment of alpha-actinin containing Z-lines and abnormal myocardial ultrastructure despite preserved cardiac function. Decreased expression of vinculin/metavinculin leads to abnormal myocyte structure without baseline physiological evidence of cardiac dysfunction. These structural changes predispose to stress-induced cardiomyopathy.

Insulin-like growth factor-II regulates PTEN expression in the mammary gland.

The tumor suppressor PTEN is altered in many cancers, including breast cancer, but only a handful of factors are known to control its expression. PTEN plays a vital role in cell survival and proliferation by regulating Akt phosphorylation, a key component of the phosphatidylinositol 3 kinase (PI3K) pathway. Here we show that insulin-like growth factor-II (IGF-II), which signals through PI3K, regulates PTEN expression in the mammary gland. IGF-II injection into mouse mammary gland significantly increased PTEN expression. Transgenic IGF-II expression also increased mammary PTEN protein, leading to reductions in Akt phosphorylation, epithelial proliferation, and mammary morphogenesis. IGF-II induced PTEN promoter activity and protein levels and this involved the immediate early gene egr-1. Thus, we have identified a novel negative feedback loop within the PI3K pathway where IGF-II induces PTEN expression to modulate its physiologic effects.