Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions.

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.

Evidence that lipoproteins are carriers of bioactive factors.

We recently demonstrated that the mitogenic effect of LDL (100 microg/mL) as well as its early intracellular signaling pathway are mediated by a pertussis-toxin (PTX)-sensitive G(i) protein-coupled receptor that is independent from its classical receptor and involves activation of extracellular response kinases (ERK1/2) (also known as p44(mapk)/p42(mapk)). In the present study we examined whether LDL-adherent factors may be responsible for some of the effects of LDL. The term "signaling activity" is used to characterize fractions that cause an increase in intracellular free Ca(2+) concentration or stimulate ERK1/2 and c-fos mRNA expression. LDL, HDL, and VLDL stimulate ERK1/2 with the following order of potency: LDL>HDL>VLDL. After delipidation of LDL with chloroform/methanol/water mixtures a PTX-sensitive signaling activity was found in one fraction arbitrarily called LDL-F. After further analysis of LDL-F compounds by high pressure liquid chromatography, a PTX-sensitive signaling activity was detected only in the fraction with a retention time of 33 minutes (arbitrarily called LDL-F33). Similarly, after separation of sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) by high pressure liquid chromatography, a PTX-sensitive signaling activity was found in the fractions 33 and 33 to 35, respectively. These findings demonstrate that the effects of LDL-F33 are mimicked by similar fractions collected from SPP/SPC, hence suggesting that these LDL-adherent molecules are possibly closely related to SPP/SPC. A PTX-sensitive signaling activity was also detected in HDL and HDL-F33. Therefore, LDL and other lipoproteins may function as carriers for bioactive phospholipids thereby contributing to the development of coronary artery disease. Our findings support a new research concept that may contribute in elucidating cellular mechanisms promoting coronary artery disease.

Identification of a phosphodiesterase I/nucleotide pyrophosphatase-related gene mRNA in rat vascular smooth muscle cells by the differential display approach.

Vascular smooth muscle cell hypertrophy and proliferation may participate in the pathophysiology of cardiovascular disease. The analysis of changes in gene expression in vascular smooth muscle cells is crucial to the understanding of the molecular biology of cardiovascular disease. An effective method for analysis of gene expression is the differential display approach. Applying the differential display approach, we identified a gp130RB13-6-related gene in vascular smooth muscle cells following stimulation with platelet-derived growth factor-BB and angiotensin II. It is well known that gp130RB13-6 is a phosphodiesterase/nucleotide pyrophosphatase. Northern blotting and reverse transcriptase-polymerase chain reaction analysis revealed a dramatic down-regulation of the gp130RB13-6-related mRNA after six hours of stimulation of the cells with both agonists. Recently, gp130RB13-6 was identified as a rat neural differentiation and tumor cell surface plasma membrane glycoprotein. These findings demonstrate that the expression of gp130RB13-6 mRNA in vascular smooth muscle cells is remarkably regulated by growth factors and therefore may play an important role in the regulation of vascular smooth muscle cell growth.

Role of mitogen-activated protein kinase in the angiotensin II-induced DNA synthesis in vascular smooth muscle cells.

The activation of mitogen-activated protein (MAP) kinase and increase in intracellular free calcium concentration ([Ca2+]i) are discussed in reference to activation of different protein kinases and growth of vascular smooth muscle cells (VSMCs). The aim of the present study was to investigate the role of angiotensin (Ang) II-induced increase in [Ca2+]i for activation of 44-kD/42-kD MAP kinase (p44mapk/p42mapk) and DNA synthesis in VSMCs. Experiments were performed by chelation of [Ca2+]i by the intracellular chelator 1,2-bis-(o-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). Ca2+ was measured by the fura 2 method. MAP kinase activation was determined by the Western blotting method. DNA synthesis was determined by measurement of [3H]thymidine incorporation into the cell DNA. Treatment of VSMCs with 20 micromol/L MAPTAM for 30 minutes resulted in a complete abolishment of the maximal Ang II-induced increase at 10 seconds. Ang II phosphorylated the p44mapk/p42mapk in a time-dependent manner, showing a maximum at 3 minutes. In MAPTAM-treated cells, the maximal phosphorylation of MAP kinase isoforms was shifted to 5 minutes, and dephosphorylation was delayed compared with untreated cells. In concordance with this finding, the induction of the MAP kinase phosphatase-1 was markedly impaired in MAPTAM-treated cells. Ang II induced a 2.3-fold increase in [3H]thymidine incorporation into DNA synthesis in untreated cells. This effect was not reduced in MAPTAM-treated cells. Treatment of the cells with PD 98059 (10 micromol/L), a MAP kinase kinase inhibitor, caused 85% inhibition of the Ang II-induced activation of MAP kinases but did not inhibit the Ang II-induced DNA synthesis. In conclusion, the Ang II-induced stimulation of the MAP kinase is a Ca2+-dependent process. Furthermore, blockade of the Ang II-induced stimulation of the early intracellular events, such as increase in [Ca2+]i or phosphorylation of the MAP kinase, is not accompanied by an inhibition of the Ang II-induced DNA synthesis.

The role of platelet-derived growth factor-BB-induced increase in cytosolic free Ca2+ in activation of mitogen-activated protein kinase and DNA synthesis in vascular smooth muscle cells.

BACKGROUND: Platelet derived growth factor (PDGF)-BB is an important vascular smooth muscle cell (VSMC) mitogen. PDGF-BB induces an increase in intracellular free calcium concentration ([Ca2+]i), an activation of mitogen-activated protein (MAP) kinase and an increase in DNA synthesis. The increase in [Ca2+]i is thought to be an important second messenger in the intracellular signalling cascade, leading to growth of VSMC. OBJECTIVE: The aim of the present study was to elucidate the role of the PDGF-BB-induced increase in [Ca2+]i in the activation of MAP kinase and increase in DNA synthesis. Binding of [Ca2+]i was performed by the intracellular chelator bis-(2-amino-5-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). METHODS: Ca2+ levels were measured by the Fura-2 method. MAP kinase activation was determined by Western blotting. DNA synthesis was determined by measurement of incorporation of [3H]-thymidine into the cell DNA. RESULTS: Administration of 50 ng/ml PDGF-BB induced an increase in [Ca2+]i, an activation of MAP kinase and an increase in DNA synthesis. In bis-(2-amino-5-methylphenoxy) ethane-N,N,N'N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM)-treated cells the PDGF-BB-induced effect on [Ca2+]i was totally blunted, whereas no effect on MAP kinase activation and DNA synthesis could be observed. CONCLUSIONS: These findings show that the effect of PDGF-BB on MAP kinase activation is independent of calcium level. [Ca2+]i might be implicated in the PDGF-BB-induced mitogenic process only in conjugation with other signalling components.