The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning-defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR-3 and PAR-6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR-3 associates with the tight-junction-associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin-1, -4 or -5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3 localization at intercellular junctions and delays ASIP/PAR-3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR-3-aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.

Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1.

We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.

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.

Gangliosides GM1, GM2 and GM3 inhibit the platelet-derived growth factor-induced signalling transduction pathway in vascular smooth muscle cells by different mechanisms.

Gangliosides appear to regulate proliferation of different cell types. In the present study, we investigated the effects of gangliosides GM1, GM2 and GM3 on platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) growth. In addition, we examined the effects of gangliosides on the PDGF-BB-dependent signalling transduction pathway in rat aortic VSMC. GM2 and GM1 inhibit the PDGF-BB-dependent receptor tyrosine autophosphorylation, stimulation of the PLC-gamma 1, increase of inositol-1,4,5-trisphosphate (InsP3), elevation in cytosolic free Ca2+ ([Ca2+]i), expression of the immediate early growth response gene c-fos and cell proliferation with the following rank order of potency GM2 > GM1. Although GM3 did not influence the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activation, it effectively inhibited the PDGF-BB-dependent InsP3 formation, [Ca2+]i and cell growth. Binding studies with 125I-PDGF-BB on VSMC in the presence and absence of 10 to 50 microM of each ganglioside revealed that GM1 and GM2 effectively inhibited the specific binding of PDGF-BB with an IC50 value of 20 microM for GM2 and 30 microM for GM1. GM3 had no significant effect on the specific 125I-PDGF-BB binding. These observations suggest that GM1 and GM2 may interact with PDGF-BB or its receptor resulting in a prevention of its binding. GM3 was able to suppress the PDGF-BB-dependent increase of InsP3 and [Ca2+]i downstream of the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activity.

Oligodeoxynucleotides directed to early growth response gene-1 mRNA inhibit DNA synthesis in the smooth muscle cell.

Vascular smooth muscle cell proliferation plays a central role in the pathophysiology of cardiovascular diseases. The induction of the early growth response gene-1 (egr-1) mRNA is associated with different cellular processes such as cell proliferation. Antisense oligodeoxynucleotides seem to provide a promising new pharmaceutical tool for effective modification of the expression of specific genes. Hence, in the present study, the effect of 15-mer antisense oligodeoxynucleotides (targeted to the initial codon region of the egr-1 mRNA) on the angiotensin II- and platelet-derived growth factor-BB-induced growth promoting effects of aortic smooth muscle cells was evaluated. Angiotensin II- and platelet-derived growth factor-BB induced egr-1 mRNA (3.4 kb) and Egr-1 protein (80 kDa) in a time- and concentration-dependent fashion. No effects of the sense and antisense oligodeoxynucleotides on the agonist-induced elevation of the egr-1 mRNA and on the Egr-1 protein could be demonstrated. However, they effectively inhibited the angiotensin II- and the platelet-derived growth factor-BB-induced DNA synthesis. Our findings provide evidence that the oligodeoxynucleotides inhibit vascular smooth muscle cell growth via nonantisense mechanism(s).

Native low-density lipoprotein (LDL) induces the expression of the early growth response gene-1 in human umbilical arterial endothelial cells.

Low-density lipoprotein (LDL) is thought to be involved in the growth of various cell types including human endothelial cells. Nevertheless, little is known about the signal transduction mechanisms underlying the growth-promoting effects of LDL in endothelial cells. Furthermore, the question whether native LDL participates in the described effects remains unanswered. Here, we show that native LDL induces a dose-dependent elevation in free intracellular Ca(2+)-concentration ([Ca2+]i) as well as a rapid and prolonged increase in intracellular pH (pHi) in human umbilical arterial endothelial cells (HUAEC). Native LDL induces a dose-dependent increase of early growth response gene-1 (egr-1) mRNA expression. The effect is maximal 30 min after addition of LDL to the culture medium. Moreover, native LDL causes an increase in DNA-synthesis and cell proliferation. In addition, the effect of acidic fibroblast growth factor (aFGF) on HUAEC proliferation was enhanced by native LDL.

Action of dihydropyridine calcium antagonists on early growth response gene expression and cell growth in vascular smooth muscle cells.

OBJECTIVE: Evidence suggests that calcium antagonists may suppress vascular smooth muscle cell (VSMC) growth and proliferation, which may be a crucial step in the pathogenesis of hypertension and atherosclerosis. DESIGN: The effects of the dihydropyridine calcium antagonists nifedipine, nitrendipine, nisoldipine, nimodipine and isradipine on cell growth induced by platelet-derived growth factor (PDGF)-AB and angiotensin II (Ang II), and expression of the transcription factors c-fos and early-growth response gene 1 (egr-1) were investigated. METHODS: Proliferation of VSMC in culture was measured by [3H]-thymidine incorporation into cell DNA and by cell count. Expression of c-fos and egr-1 messenger RNA (mRNA) was determined by the Northern blot technique. RESULTS: All of the calcium antagonists blunted the PDGF-induced rise in VSMC DNA synthesis. The inhibitory potency of isradipine on PDGF-stimulated DNA synthesis was approximately 10-fold that of the other calcium antagonists used, isradipine having a half-maximal inhibitory concentration (IC50) of (4.2 +/- 0.16) x 10(-7) mol/l. The calcium antagonists investigated also inhibited Ang II-induced DNA synthesis. Isradipine (10(-6) mol/l) completely abolished the PDGF-induced cell proliferation. Both PDGF (50 ng/ml) and Ang II (10(-7) mol/l) induced c-fos and egr-1 mRNA expression, having maximum effect after 30 min. In the case of c-fos, pre-incubation with 5 x 10(-6) mol/l isradipine led to a decrease in both Ang II- and PDGF-induced expression of this immediate-early gene. The expression of egr-1 was not affected by pre-incubation with 5 x 10(-6) mol/l isradipine. CONCLUSIONS: All calcium antagonists investigated in the present study inhibited cell growth. Isradipine was more potent in blocking growth factor-induced cell growth than the other calcium antagonists studied. The inhibitory effect of the dihydropyridine calcium antagonists appears to be dependent on the expression of c-fos.

The induction of early response genes in rat smooth muscle cells by PDGF-AA is not sufficient to stimulate DNA-synthesis.

The effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB and BB on the induction of the early growth response genes c-fos, egr-1 and c-myc mRNA in vascular smooth muscle cells from rat was compared with their respective mitogenic potency. The three PDGF isoforms strongly stimulated the induction to a similar extent. In contrast, PDGF-AB and -BB provoked a marked DNA synthesis whereas PDGF-AA exerted only a poor mitogenic effect in smooth muscle cells. PDGF-AA-stimulated receptor autophosphorylation was not detectable in comparison with the strong effect elicited by PDGF-AB or -BB and correlated with its low mitogenicity but not with the almost equal induction of the early response genes. It is discussed that no or only very low receptor phosphorylation is required to link receptor activation to the induction of c-fos, egr-1 or c-myc. Furthermore the induction of the investigated gene does not seem to be sufficient for an optimal mitogenic response.

EXP3174, a metabolite of losartan (MK 954, DuP 753) is more potent than losartan in blocking the angiotensin II-induced responses in vascular smooth muscle cells.

OBJECTIVE: EXP3174 is a metabolite of losartan (previous name DuP753), which is a non-peptide angiotensin II receptor antagonist. DESIGN: The inhibitory potency of these two antagonists on the angiotensin II-induced responses in vascular smooth muscle cells (VSMC) was investigated. METHODS: The effect of angiotensin II on cell growth was determined by [3H]-thymidine incorporation into cell DNA and by cellular protein measurements. Intracellular cytosolic Ca2+ concentration was measured by the fura-2 method. Inositolphosphates were determined by high-performance liquid chromatography after cell labelling with myo-[2-3H]-inositol. The early growth response gene-1 (Egr-1) messenger RNA (mRNA) expression was determined by the Northern blotting method. Binding and displacement studies of the antagonists were performed using [125I]-angiotensin II. RESULTS: An apparent dissociation constant (Kd) of 5.9 nmol/l for [125I]-angiotensin II (maximal binding coefficient 69 fmol/10(6) cells) was found. The specific binding of [125I]-angiotensin II to VSMC was inhibited by losartan, EXP3174 and saralasin with a half-maximal inhibitory concentration (IC50) of 1.0 X 10(-8), 1.1 X 10(-9) and 1.8 X 10(-9) mol/l, respectively. EXP3174 and losartan abolished the angiotensin II-induced formation of inositolphosphates in VSMC. EXP3174 and losartan inhibited the angiotensin II-induced elevation of intracellular cytosolic Ca2+ concentration with an IC50 of 5 X 10(-9) and 5 X 10(-8) mol/l, respectively. EXP3174 was more effective than losartan in blocking the angiotensin II-induced increase in Egr-1 mRNA. EXP3174 and losartan inhibited the angiotensin II-induced cell protein synthesis with an IC50 of 3 X 10(-9) and 4 X 10(-8) mol/l, respectively. CONCLUSIONS: These results indicate that EXP3174 is significantly more potent than losartan in blocking angiotensin II-induced cellular responses.