Inhibition of Cx43 attenuates ERK1/2 activation, enhances the expression of Cav­1 and suppresses cell proliferation.

In addition to being an important component of the gap junction, connexin 43 (Cx43) has been shown to regulate other cellular functions, including cell proliferation. This regulatory role of Cx43 may be important in therapeutic situations, including wound healing or ischemic injuries. Caveolin­1 (Cav­1) has been shown to regulate angiogenesis. The aim of the present study was to analyze whether Cx43 counter­regulates Cav­1 in controlling the proliferation and migration of endothelial cells. The inhibition of Cx43 with niflumic acid, flufenamic acid and 18­α­glycyrrhetinic acid in cultured human umbilical vein endothelial cells resulted in decreased phosphorylation of extracellular signal­regulated kinase (ERK)1/2 and increased expression of Cav­1, as shown by western blot analysis. Furthermore, the inhibition of Cx43 resulted in a 50±7% decrease in cell proliferation, determined using a crystal violet assay, a 48±5% decrease in migration, determined using a migration assay, and a 49±6% decrease in endothelial tube formation, determined using a Matrigel assay, compared with the control. Similar results were obtained following specific inhibition of Cx43 by mimetic peptides (Gap26 and Gap27). Inhibition of the mitogen­activated protein kinase kinase/ERK pathway with PD­98059 resulted in an increased expression of Cav­1 and a reduction in the expression of Cx43. Furthermore, cell proliferation, migration and tube formation in endothelial cells were impaired. By contrast, downregulation of the protein expression of Cav­1 by small interference RNA resulted in increased expression of Cx43 and phosphorylation of ERK1/2. Accordingly, the number of cells in the Cav­1 treated­group increased by 35±5% compared with the controls. The data of the present study showed that Cav­1 suppressed cell proliferation by inhibiting the activity of Cx43, which is upstream of ERK1/2. The downregulation of Cav­1 protein resulted in loss of the inhibitory activity of Cav­1 on cell proliferation and led to increased cell proliferation. This counter­regulatory effect of Cx43 may be of importance in therapeutic angiogenesis.

Effects of nicotine on PTHrP and PTHrP receptor expression in rat coronary endothelial cells.

AIMS: The study was aimed to investigate whether nicotine affects endothelial expression of PTHrP and PTHrP receptor, a peptide system involved in endothelial protection against apoptosis. METHODS: Isolated and cultured rat coronary endothelial cells were used. Immunoblot techniques were used to study activation of mitogen-activated protein (MAP) kinases and to quantify PTHrP and PTHrP receptor expression. Real-time RT-PCR was used to quantify PTHrP, PTHrP-receptor, bcl-2, and bax mRNA expression. The rate of apoptosis was determined by HOE33258 staining and confirmed by quantification of the bcl-2-to-bax ratio. In vitro data were compared to hearts from rats exposed to cigarette smoking. RESULTS: Nicotine induced PTHrP protein expression at nanomolar levels and small increases of PTHrP release (≈8%). Antagonists directed against the α7 subunit of cholinergic receptors, the most prominent isoform, attenuated nicotine-dependent increases of PTHrP expression. This effect of nicotine was p38 MAPK dependent. Nicotine at micromolar concentrations reduced PTHrP receptor expression. In vitro and in vivo we found a correlation between PTHrP receptor expression and bcl-2 expression. CONCLUSION: Nicotine induces PTHrP expression in endothelial cells but excessive concentrations of nicotine reduce PTHrP receptor expression thereby attenuating any protective effects of PTHrP against apoptosis.

Parathyroid hormone-related protein (PTHrP)-dependent regulation of bcl-2 and tissue inhibitor of metalloproteinase (TIMP)-1 in coronary endothelial cells.

BACKGROUND: An increased susceptibility of micro-vascular endothelial cells to apoptosis is considered to be an initial event leading to atherosclerosis. Parathyroid hormone-related peptide (PTHrP) is known to protect endothelial cells against apoptosis by the regulation of the anti-apoptotic gene bcl-2. As tissue inhibitor of metalloproteinase (TIMP-1) expression is regulated by bcl-2, we hypothesized that endothelial expression of PTHrP also regulates the expression of TIMP-1. METHODS: The steady state mRNA expressions of bcl-2, bax, TIMP-1, and TIMP-2 were analyzed by real-time RT-PCR and their protein expression by immunoblotting. The tissue distribution of PTHrP was investigated in cryosections of hearts from normotensive and hypertensive rats. RESULTS: Phenylephrine, an alpha(1)-adrenoceptor agonist, increased the expression of PTHrP, bcl-2, and TIMP-1. Transfection of endothelial cells with oligonucleotides directed against PTHrP attenuated this effect. Antisense transfection and TGF-beta(1) (10 ng/ml) decreased the expression of PTHrP, bcl-2, TIMP-1, and TIMP-2, but not that of bax. Endothelial cells were identified as the main source of PTHrP in the heart. Endothelial cells in hearts from spontaneously hypertensive rats showed reduced staining with a PTHrP antibody compared to control normotensive hearts. CONCLUSIONS: These data suggests that the down-regulation of PTHrP favours atherosclerosis in chronic pressure overload.

Parathyroid hormone-related protein (PTHrP) signal cascade modulates myocardial dysfunction in the pressure overloaded heart.

BACKGROUND: Pressure overload induces the cardiac expression of parathyroid hormone-related protein (PTHrP). Plasma levels are elevated in patients with heart disease. It is unknown whether this represents an epiphenomenon or suggests involvement in hypertrophy. AIM: To identify a potential role of PTHrP in pressure induced hypertrophy and heart failure. METHODS AND RESULTS: Pressure load was produced via thoracic aortic constriction (TAC) and application of a PTHrP antagonist (PTHrP(7-34)) via osmotic minipumps in mice. Main findings were confirmed in vitro by exposing isolated adult ventricular mice cardiomyocytes to PTHrP(1-34) (100 nmol/l). TAC treated animals developed myocardial hypertrophy within 2 weeks. The heart weight to body weight ratio increased from 5.02+/-0.14 mg/g (sham/vehicle) and 5.16+/-0.19 mg/g (sham/antagonist) to 6.59+/-0.85 mg/g (TAC/vehicle) and 7.07+/-0.80 mg/g (TAC/antagonist) (each n=6-8; p<0.05 for TAC vs. sham; not significantly different between TAC groups). In parallel, the expression of atrial natriuretic factor increased. Cardiac dysfunction (+dP/dt, -dP/dt), however, was significantly lower in TAC mice receiving the antagonist, and SERCA2 expression was higher. Isolated cardiomyocytes exposed to PTHrP(1-34) developed reduced cell shortening. This reduction in cell function was abolished in the co-presence of the antagonist. CONCLUSION: PTHrP contributes to the progression of cardiac dysfunction in the pressure overloaded heart.

PspGI, a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes.

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.