Angel Pascual

3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation

The mechanism by which 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) induce apoptosis in vascular smooth muscle cells (VSMCs) is unknown. In this work, we demonstrate that treatment of VSMCs with simvastatin and atorvastatin inhibited Bcl-2 expression in a time and dose-dependent manner, while Bax expression was not modified. This effect was reversed by mevalonate (100 micromol/l), farnesylpyrophosphate (5 micromol/l) or geranylgeranylpyrophosphate (5 micromol/l), suggesting the involvement of protein prenylation. The treatment of VSMCs with lipophilic statins was associated with decreased prenylation of p-21 Rho A and mevalonate, farnesyl pyrophosphate (F-PP) and geranylgeranyl pyrophosphate (G-PP) reversed prenylation to basal levels. In addition, overexpression of constitutively active Q63L Rho A prevented, at least in part, apoptosis induced by statins and downregulation of Bcl-2. We also investigated the participation of caspases (proteases) in the apoptosis induced by statins. The treatment of VSMCs with lipophilic statins induced activation of the caspase 9, the first caspase of the mitochondrial pathway. Coincubation of VSMCs with the caspase inhibitor ZVAD-fmk (100 micromol/l) significantly inhibited lipophilic statin-induced apoptosis. These findings indicate that the downregulation of Bcl-2 by Rho GTPases mediates statin-induced apoptosis and suggest a new potential mechanism of action for these drugs on the regulation of cell number in the atherosclerotic lesions.

Thyroid hormone receptors, cell growth and differentiation

BACKGROUND Tissue homeostasis depends on the balance between cell proliferation and differentiation. Thyroid hormones (THs), through binding to their nuclear receptors, can regulate the expression of many genes involved in cell cycle control and cellular differentiation. This can occur by direct transcriptional regulation or by modulation of the activity of different signaling pathways. SCOPE OF REVIEW In this review we will summarize the role of the different receptor isoforms in growth and maturation of selected tissues and organs. We will focus on mammalian tissues, and therefore we will not address the fundamental role of the THs during amphibian metamorphosis. MAJOR CONCLUSIONS The actions of THs are highly pleiotropic, affecting many tissues at different developmental stages. As a consequence, their effects on proliferation and differentiation are highly heterogeneous depending on the cell type, the cellular context, and the developmental or transformation status. Both during development and in the adult, stem cells are essential for proper organ formation, maintenance and regeneration. Recent evidence suggests that some of the actions of the thyroid hormone receptors could be secondary to regulation of stem/progenitor cell function. Here we will also include the latest knowledge on the role of these receptors in proliferation and differentiation of embryonic and adult stem cells. GENERAL SIGNIFICANCE The thyroid hormone receptors are potent regulators of proliferation and differentiation of many cell types. This can explain the important role of the thyroid hormones and their receptors in key processes such as growth, development, tissue homeostasis or cancer. This article is part of a Special Issue entitled Thyroid hormone signalling.

Comparison of the effects of forskolin and dibutyryl cyclic AMP in neuroblastoma cells: evidence that some of the actions of dibutyryl cyclic AMP are mediated by butyrate.

Abstract: We have compared the effects of forskolin, N6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate (dibutyryl cyclic AMP, Bt2‐cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2‐cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose‐dependent inhibition of cell proliferation, but the effect of Bt2‐cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2‐cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2‐cAMP than in untreated cells or in forskolin‐treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2‐cAMP produced histone acetylation, a well‐known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2‐cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1°, a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1° in the cells treated with Bt2‐cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2‐cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.

Thyroid Hormones Regulate β-Amyloid Gene Splicing and Protein Secretion in Neuroblastoma Cells1

The β-amyloid protein (Aβ), the major component of the senile plaques found in Alzheimer brains, derives from a larger β-amyloid precursor protein (APP). Alternative splicing of the APP gene yields three major APP messenger RNAs (mRNAs), which, in turn, give rise to the APP770, APP751, and APP695 protein isoforms. In this study we examined the effects of thyroid hormone on APP expression in N2a-β neuroblastoma cells. T3 caused a significant increase in the APP770 mRNA band, in detriment of the APP695 mRNA, which was proportionately reduced. In agreement with these results, T3 markedly altered the relative ratio of intracellular APP isoforms, increasing the amount of APP770 and causing an equivalent reduction of the immature APP695 isoform. In accordance with these results, the soluble APP695-derived form was specifically reduced in the culture medium obtained from T3-treated cells. In contrast, the increase in intracellular APP770 was not followed by an enhanced release of soluble derivatives of this isof...

Nerve growth factor and ras regulate β-amyloid precursor protein gene expression in PC12 cells

Abstract: The β‐amyloid protein, the major component of the vascular and plaque amyloid deposits that characterize Alzheimers disease, derives from a larger β‐amyloid precursor protein (APP) that is expressed in both neural and nonneural cells. An increased expression of APP might actively contribute to the development of the pathology; however, the mechanisms involved in the regulation of APP gene expression are not yet well understood. In PC12 cells, a rat pheochromocytoma cell line, we have demonstrated that nerve growth factor (NGF) induces the APP gene expression and increases APP mRNA levels in the presence of 0.5 or 15% serum. Expression of activated ras in the PC12 cell subline UR61 also leads to a significant increase in content of APP transcripts, and a dominant negative mutant of ras blocks the NGF‐induced response. Other ligands of tyrosine kinase receptors, such as fibroblast growth factor, which causes morphological differentiation, or epidermal growth factor, which induces cell growth, also increase APP mRNA levels in PC12 cells. These results suggest that ras mediates the induction of APP gene expression by NGF and other ligands of tyrosine kinase receptors.

Nerve growth factor modulates the expression and secretion of β‐amyloid precursor protein through different mechanisms in PC12 cells

The β‐amyloid protein, component of the senile plaques found in Alzheimer brains is proteolytically derived from the β‐amyloid precursor protein (APP), a larger membrane‐associated protein that is expressed in both neural and non‐neural cells. Overexpression of APP might be one of the mechanisms that more directly contributes to the development of Alzheimers disease. The APP gene expression is regulated by a number of cellular mediators including nerve growth factor (NGF) and other ligands of tyrosine kinase receptors. We have previously described that NGF increases APP mRNA levels in PC12 cells. However, the molecular mechanisms and the precise signalling pathways that mediate its regulation are not yet well understood. In the present study we present evidence that NGF, and to a lesser extent fibroblast growth factor and epidermal growth factor, stimulate APP promoter activity in PC12 cells. This induction is mediated by DNA sequences located between the nucleotides − 307 and − 15, and involves activation of the Ras–MAP kinase signalling pathway. In contrast, we have also found that NGF‐induced secretion of soluble fragments of APP into the culture medium is mediated by a Ras independent mechanism.

Brain-derived neurotrophic factor stimulates β-amyloid gene promoter activity by a Ras-dependent/AP-1-independent mechanism in SH-SY5Y neuroblastoma cells

The β‐amyloid peptide, the major component of Alzheimer‐associated plaques, derives from a larger β‐amyloid precursor protein (APP), that is expressed in both neural and non‐neural cells. Overexpression of APP actively contributes to the development of senile plaques and is considered a risk factor for the disease. APP expression is regulated by a variety of cellular mediators, among them ligands of tyrosine kinase receptors. In this study, we present evidence that brain‐derived neurotrophic factor (BDNF) modulates, in a dose‐ and time‐dependent fashion, APP promoter activity in SH‐SY5Y neuroblastoma cells transiently expressing the receptor TrkB. The APP promoter contains two potential AP‐1 sites, and we examined whether or not protein kinase C (PKC) and the AP‐1 sites of the promoter mediate the BDNF‐induced stimulation of APP. Stimulation of APP promoter activity by BDNF was not affected by the PKC inhibitor bisindolylmaleimide, or by dominant negative mutants of the AP‐1 components Fos and Jun, which, however, blocked the response to phorbol esters. These results suggest that activation of the APP promoter by BDNF is largely independent of PKC and AP‐1. In contrast, activated Ras increased APP promoter activity in SH‐SY5Y cells, and a dominant negative mutant of Ras abolished BDNF‐mediated promoter stimulation. Taken together, our results suggest a mechanism that involves activation of the Ras/MAP kinase signaling pathway, and phosphorylation of as yet unidentified effectors which in turn can activate response elements within the APP promoter.

The thyroid hormone receptor β induces DNA damage and premature senescence

Thyroid hormone and its receptor act in concert with NRF1 to increase cellular respiration and reactive oxygen species production, leading to DNA damage and premature senescence in susceptible cells.

Triiodothyronine (T3) induces neurite formation and increases synthesis of a protein related to MAP 1B in cultured cells of neuronal origin.

Neuroblastoma (N2A) cells were found to develop axon-like neurite extensions when grown in the presence of triiodothyronine (T3), while C6 cells (of glial origin) did not. Analysis of radiolabelled protein synthesis showed that, in N2A only, T3 increased the synthesis of a polypeptide corresponding in electrophoretic mobility to the microtubule-associated protein MAP 1B. Immunoblotting of total cell proteins with a monoclonal antibody confirmed that this polypeptide was immunologically related to MAP 1B. Further studies using indirect immunofluorescence with monoclonal antibodies against both tubulin and MAP 1B showed that both antigens were present in neurites. Taken together, these results suggest that T3 may control maturation of neural tissue via effects on the microtubule-associated proteins in cells of neuronal origin.

Down-regulation of thyroid hormone nuclear receptor levels by l-triiodothyronine in cultured glial C6 cells ☆

L-Triiodothyronine (T3) produced a time- and dose-dependent depletion of nuclear thyroid hormone receptor levels in C6 cells, a rat glioma cell line. Receptor number diminished by 30-40% after a 48 h incubation with concentrations of T3 that saturate the nuclear receptor. The nuclear binding curve obtained in cells incubated for 48 h with T3 was shifted leftward of the curve obtained after a 3 h incubation, which indicates an apparent increase in receptor affinity after long-term incubation with T3. However, this change probably represents a further equilibration of the hormone, since the dissociation rate from the nuclei was similar in C6 cells after long- and short-term incubation with T3. The effect of T3 was further demonstrated in C6 cells incubated with short-chain fatty acids. Butyrate and isobutyrate increased receptor levels, and T3 partially decreased the response to these compounds. These findings suggest the existence of a desensitization process by which C6 glial cells would be protected against an excess of thyroid hormone.