TGF-beta1 is increased in a transgenic mouse model of familial Alzheimer's disease and causes neuronal apoptosis.

Alzheimer's disease (AD) is a neurodegenerative disorder, due to excess amyloid-beta peptide (Abeta). TGF-beta1 and beta-catenin signaling pathways have been separately implicated in modulating Abeta-neurotoxicity. However, the underlying mechanisms remain unclear. Here, we report that TGF-beta1 and nuclear Smad7 and beta-catenin levels were markedly upregulated in cortical brain regions of the TgCRND8 mice, a mouse model of familial Alzheimer's disease. Coimmunoprecipitation of cortical brain tissue lysates revealed an interaction between Smad7 and beta-catenin. This interaction which was significantly enhanced in the TgCRND8 mice was also associated with an increase in TCF/LEF DNA-shift binding activity. TCF/LEF reporter gene activity was significantly increased in mouse primary cortical neuronal cultures (MCN) from the TgCRND8 mice, compared to controls. Interestingly, exposure of MCN to Abeta(1-42) led to an increase in TGF-beta1 and nuclear levels of both beta-catenin and Smad7. Furthermore, addition of TGF-beta1 to the MCN caused an increase in apoptosis and Smad7 levels. When Smad7 or beta-catenin levels were reduced by siRNA, TGF-beta1-induced apoptosis was suppressed, indicating that both Smad7 and beta-catenin are required for TGF-beta1-induced neurotoxicity. Since Abeta(1-42)-induced TGF-beta1, we suggest that TGF-beta1 may amplify Abeta(1-42)-mediated neurodegeneration in AD via Smad7 and beta-catenin interaction and nuclear localization.

Lovastatin protects human neurons against Abeta-induced toxicity and causes activation of beta-catenin-TCF/LEF signaling.

Alzheimer's disease (AD) is characterized by cognitive decline due to excess amyloid beta peptide (Abeta), neurofibrillary tangles, and neuronal loss. Abeta promotes neuronal apoptosis in AD by activating glycogen synthase kinase-3beta (GSK-3beta), leading to degradation of beta-catenin and inactivation of Wnt signaling. beta-Catenin interacts with the T-cell factor (TCF)/Lymphoid enhancer factor (LEF)-nuclear complex to mediate Wnt signaling and cell survival. Statins are associated with decreased prevalence of AD. Lovastatin has been shown to decrease the production of Abeta and to promote neuronal survival. The mechanisms of how statins promote neuronal survival are unclear. We propose that the neuroprotective effect of lovastatin may be due to inactivation of GSK-3beta activity, resulting in induction of Wnt signaling. Here, we report that lovastatin prevented Abeta-induced apoptosis in human SK-NSH cells. This was accompanied by reduction in active GSK-3beta, and increased nuclear translocation of beta-catenin, TCF-3, and LEF-1. Lovastatin treatment induced an increase in TCF/LEF-chloramphenicol acetyl transferase (CAT) gene reporter activity. More importantly, beta-catenin and TCF were required for the neuroprotective function of lovastatin. Our results suggest that lovastatin protects neuronal cells from Abeta-induced apoptosis and causes reduction in GSK-3beta activity, resulting in activation of Wnt signaling.

The role of transforming growth factor beta signaling in messenger RNA stability.

Transforming growth factor beta (TGF-beta) is a biologically multipotent regulatory protein implicated in functions that include the regulation of cellular growth, differentiation, extracellular matrix formation, and wound healing. It also plays a role in the pathologies of Alzheimer's disease, cancer and autoimmune disorders. TGF-beta modulates gene expression by affecting transcriptional activation and mRNA turnover rate. Steady-state mRNA levels depend on both the transcriptional activity and mRNA half-life. The stability of mRNA can be modified by the binding of trans-acting factors to cis-elements on the message. These can protect the mRNA from cleavage by RNAses, or they may promote mRNA cleavage. Changes in mRNA stability can lead to changes in the proteome and subsequently in cellular metabolism. The SMAD family of proteins has been implicated in the transduction of the TGF-beta signal, where they regulate transcriptional activity. This review attempts to provide new insights into the role played by TGF-beta in the regulation of mRNA turnover.

Myogenic signaling by lithium in cardiomyoblasts is Akt independent but requires activation of the beta-catenin-Tcf/Lef pathway.

Rat H9c2 cardiomyoblasts can proliferate and maintain an undifferentiated state in the presence of serum. These cardiomyoblasts have been used as a cellular model to study myogenic differentiation after serum withdrawal. Here, we examined the effects of lithium, a known inhibitor of glycogen synthase kinase-3beta and activator of Wnt pathway in myogenic differentiation. We show that in the presence of serum, lithium induced the differentiation of H9c2 cells as measured by multinucleated myotube formation and expression of the muscle-specific proteins, myogenin and skeletal alpha-actin. This differentiation was preceded by nuclear accumulation of beta-catenin, which was associated with increased Tcf/Lef-dependent transcription. We also observed that lithium mediated the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target Akt. Inhibition of PI3-kinase by LY294002 and over-expression of dominant-negative PI3-kinase caused a marked reduction in beta-catenin levels. This inhibition was associated with decreased beta-catenin-Tcf/Lef-dependent transcription, lack of multinucleated myotube formation, and expression of the muscle-specific proteins. In contrast, expression of dominant-negative Akt failed to inhibit the effects of lithium. We conclude that the capacity of lithium to overcome the inhibitory effects of serum and to induce the differentiation of H9c2 cardiomyoblasts is mediated, in part, by the stabilization and nuclear translocation of beta-catenin in a PI3-kinase-dependent but Akt-independent manner. Once activated, beta-catenin then interacts with the Lef/Tcf complex to regulate expression of myogenic-inducing genes.

Cellular signaling pathways affect the function of ribonucleotide reductase mRNA binding proteins: mRNA stabilization, drug resistance, and malignancy (Review).

Ribonucleotide reductase is an enzyme that is essential for DNA synthesis and repair. It is composed of 2 dimeric proteins called R1 and R2 that are both necessary for enzymatic activity that reduces ribonucleotides to deoxyribonucleotides. This is the rate-limiting reaction that provides a supply of precursors for DNA synthesis therefore it is essential for cell proliferation. The importance of understanding the complex regulation of ribonucleotide reductase is emphasized by observations that mechanisms controlling its expression and activity may be altered during malignant cell proliferation which leads to drug resistance, making it a useful target to develop chemotherapeutic compounds in the treatment of cancer. Expression studies with the R1 and R2 genes have provided evidence for a direct role for the components of ribonucleotide reductase in determining malignant potential. Ribonucleotide reductase is regulated by transcriptional activation of gene expression and post-transcriptional mechanisms that alter mRNA message stability. Post-transcriptional regulation of mRNA turnover plays an important role in modulating mRNA steady state levels and therefore directly influences gene expression. The 3'-untranslated region (UTR) of R1 and R2 messages contain sequences that are important in regulating gene expression through changes in message stability. Studies have found that mRNA message stability is mediated by growth factors, cytokines and tumor promoters. Several studies have elucidated signal transduction pathways of tumor promoters, TGF-beta and oxidation/reduction agents. This report reviews how knowledge of these signaling pathways is revealing new insights into how ribonucleotide reductase mRNA binding proteins are important in regulating cellular proliferation, drug resistance and malignancy.

Late Simian virus 40 transcription factor is a target of the phosphoinositide 3-kinase/Akt pathway in anti-apoptotic Alzheimer's amyloid precursor protein signalling.

The association of familial Alzheimer's disease (FAD) with mutations in Alzheimer's amyloid precursor protein (APP) suggests important functions for APP in the central nervous system. Mutations in APP impair its function to confer resistance to apoptosis in cells under stress, and this may contribute to neurodegeneration in Alzheimer's disease (AD) brain, but the mechanisms involved are unknown. We examined the role of the late Simian virus 40 transcription factor (LSF), in anti-apoptotic APP pathways. We show that in APP-deficient B103 cells, expression of wild-type human APP (hAPPwt), but not of FAD-mutant APP, inhibited staurosporine (STS)-induced apoptosis. This inhibition was further enhanced by expression of LSFwt, although LSFwt alone was not sufficient to inhibit STS-induced apoptosis. In contrast, expression of dominant-negative LSF led to a marked increase in STS-induced cell death that was significantly blocked by hAPPwt. These effects of APP were accompanied by LSF nuclear translocation and dependent gene transcription. The activation of LSF is dependent on the expression of hAPPwt and is inhibited by the expression of dominant-negative forms of either phosphoinositide 3-kinase or Akt. These results demonstrate that LSF activation is required for the neuroprotective effects of APP via phosphoinositide 3-kinase/Akt signalling. Alterations in this pathway by aberrations in APP and/or LSF could promote neuronal loss in AD brain, due to secondary insults. Thus a link is established between APP and LSF and AD.

Anti-apoptotic wild-type Alzheimer amyloid precursor protein signaling involves the p38 mitogen-activated protein kinase/MEF2 pathway.

Alzheimer amyloid precursor protein (APP) effectively protects against apoptosis in neuronal cells under stress, but the mechanisms of this anti-apoptotic effect remain largely unknown. Transcription factors act as critical molecular switches in promoting neuronal survival. The myocyte enhancer factor-2 (MEF2) is a transcription factor, and is known to be necessary for neurogenesis and activity-dependent neuronal survival. This study examined the possible role of MEF2 in the anti-apoptotic signaling pathways activated by APP. We report that expression of wild-type human APP (hAPPwt) but not familial Alzheimer's disease mutant APP (FAD-hAPPmut) in APP-deficient rat B103 cells led to a significant increase in the level of phosphorylated MEF2. This differential phosphorylation was dependent on enhanced activation of p38 mitogen-activated protein kinase (p38 MAPK). Also, expression of hAPPwt mediated an increase in MEF2 DNA binding affinity that correlated with p38 MAPK-dependent trans-activation of a MEF2-responsive reporter gene. Furthermore, over-expression of dominant negative MEF2 in hAPPwt-expressing cells enhanced staurosporine-induced apoptosis, in contrast MEF2wt enhanced the capacity of hAPPwt to confer resistance to apoptosis. Thus, MEF2 plays a critical role in APP-mediated signaling pathways that inhibit neuronal apoptosis. A model of anti-apoptotic APP signaling is proposed where APP mediates p38 MAPK-dependent phosphorylation and activation of MEF2. Once activated MEF2 regulates neuronal survival by stimulation of MEF2-dependent gene transcriptions. Alteration of this function by mutations in APP and aberrant APP processing could contribute to neuronal degeneration seen in AD.

Transcriptional activation and increase in expression of Alzheimer's beta-amyloid precursor protein gene is mediated by TGF-beta in normal human astrocytes.

The overexpression of the Alzheimer amyloid precursor protein (APP) and its subsequent proteolytic processing may be one of several factors contributing to amyloid beta-peptide (Abeta) deposition in plaques and microvasculature in Alzheimer's disease (AD) brain. Cytokines and growth factors can influence the expression of APP in response to brain injury, but the underlying mechanisms are largely unknown. We examined the mechanisms by which transforming growth factor-beta (TGF-beta) affects the expression of APP in normal human astrocytes. We report that, TGF-beta up-regulated the expression of APP at the transcription level as determined by nuclear run-on experiments. Transient transfection of astrocytes with APP gene promoter (-2832 bp) chloramphenicol acetyltransferase (CAT) reporter constructs led to increased reporter activity upon TGF-beta stimulation. This reporter activity was mainly attributed to the APP proximal domain (-488 bp). The increase in APP gene transcription was associated with significant accumulation of intracellular APP, APP carboxyl terminal derived fragments, and total secreted Abeta. In addition, we observed a significant increase in levels of TGF-beta in Abeta plaques and its immediate vicinity in AD-affected brain relative to controls. These results indicate that high levels of TGF-beta in the cortex, may serve to up-regulate APP synthesis in reactive astrocytes and indirectly contributes to Abeta deposition. Closely related processes may induce cerebrovascular pathology in AD brain.

Transforming growth factor-beta-induced transcription of the Alzheimer beta-amyloid precursor protein gene involves interaction between the CTCF-complex and Smads.

Transforming growth factor-beta-1 (TGF-beta), a key regulator of the brain responses to injury and inflammation, has been implicated in upregulating the expression of the Alzheimer amyloid precursor protein (APP) and Alzheimer's disease (AD) pathogenesis. However, little is known about the mechanisms underlying the effects of TGF-beta on APP expression. Analysis of APP promoter activity upstream of the chloramphenicol acetyltransferase reporter gene in normal human astrocytes (NHAs), revealed that the APP promoter binding beta (APBbeta) site (-93/-82) is responsive to TGF-beta. This site interacts with the zinc finger nuclear factor CTCF, involved in APP transcriptional activity. As determined by gel shift assay, there was no significant difference in the CTCF-APBbeta complex binding activity in the presence or absence of TGF-beta treatment of NHAs. To further investigate the contributions of the CTCF-complex and Smad proteins to the TGF-beta induced APP promoter activity, we examined the distribution of these factors and their DNA binding activity. Interestingly, upon TGF-beta treatment both Smads 3 and 4 were translocated to the nuclei in contrast to Smad 2, which was cytoplasmic. However, CTCF was predominantly localized in the nuclei irrespective of TGF-beta treatment. Gel super shift assay coupled with Western blot analysis showed that Smads 3 and 4 specifically associated with the CTCF-APBbeta complex. In addition, AD brain sections showed increased expression and nuclear localization of Smad 4, which correlated with higher levels of APP and TGF-beta. However, over expression of Smad 4 on its own was not sufficient to affect APP expression. These results demonstrate that TGF-beta activation of Smad protein complexes promotes transcription of the APP gene. Increased synthesis of APP may in part determine Abeta production and deposition in affected AD brain.