Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Breton, Michelyne; Berrou, Eliane; Mc, Brahimi-Horn; Deudon, Elisabeth; Picard, Jacques

doi:10.1016/0014-4827(86)90487-8

Cited by 32 publications

(22 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell Culture and Isolation of VSMC Nuclei-VSMCs were prepared from the thoracic aortas of pigs 6 weeks of age using the explant technique and cultured as described previously (27). For all experiments, VSMCs were used from the second to the fifth passage.…”

Section: Methodsmentioning

confidence: 99%

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

Déléris

Bacqueville

Gayral

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 )-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P 3 phosphatases. Therefore, we investigated the dephosphorylation of [ 32 P]PtdIns(3,4,5)P 3 specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P 3 phosphatase assays revealed the production of both [ 32 P]PtdIns(3,4)P 2 and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P 3 5-and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P 3 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [ 32 P]PtdIns(3,4,5)P 3 dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P 3 into PtdIns(3,4)P 2 within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P 3 phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.Vascular smooth muscle cell (VSMC) 1 migration and proliferation are key factors in the development of atherosclerosis, post-angioplasty restenosis, and transplant vasculopathy (1, 2). We showed previously that phosphoinositide 3-kinase (PI3K) is essential for the VSMC cycle (3), and this enzyme was implicated recently in restenosis (4). PI3Ks make up a large family of lipid kinases, but class I PI3Ks are the most well known (5-7). They convey both growth and survival signals from activated receptors; class IA PI3K (␣, ␤, and ␦) is regulated by receptors with intrinsic or associated-tyrosine kinase activity, whereas the class IB or PI3K␥ is activated by G protein-coupled receptors. PI3Ks catalyze phosphorylation of phosphoinositides (PIs) at the D-3 position of the inositol ring to generate two potent second messengers, phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), which regulate downstream effectors such as the serine/threonine protein kinases PKB (also called Akt) and PKC , and control many cellular responses involved in neointimal hyperplasia and restenosis, including cell proliferation, migration, and survival (8, 9).Because 3-phosphoinositides (3-PIs) are not hydr...

show abstract

Section: Methodsmentioning

confidence: 99%

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

Déléris

Bacqueville

Gayral

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Cell Culture and Isolation of VSMC Nuclei-VSMCs were prepared from 6-week-old pig thoracic aorta using the explant technique (26) and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, as previously described (27). For all the experiments, VSMCs were used from the third to the sixth passage.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a G Protein-activated Phosphoinositide 3-Kinase in Vascular Smooth Muscle Cell Nuclei

Bacqueville

Déléris

Mendre

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5-(␥-thio)triphosphate (GTP␥S) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), whereas guanosine 5-(␤-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTP␥S-induced PtdIns(3,4,5)P 3 synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P 3 accumulation, suggesting that nuclear G i /G 0 proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of G␣ 0 proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85␣ subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3K␥. These results indicate the existence of a G proteinactivated PI3K inside VSMC nucleus that might be involved in the contol of VSMC proliferation and in the pathogenesis of vascular proliferative disorders. Vascular smooth muscle cells (VSMCs)1 play a central role in the fibroproliferative response during the development of atherosclerosis and of restenosis after angioplasty (1, 2). Recently, we have demonstrated that phosphoinositide 3-kinase (PI3K) was essential for the progression of VSMCs throughout the G 1 phase of the cell cycle (3), implying that a better understanding of the PI3K signaling pathway might be of pathophysiological relevance. PI3K phosphorylates the D3 position of the inositol ring in phosphoinositides (PI) to generate the putative second messengers PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 (4). In addition to proliferation, the PI3K products have also been involved in cell transformation, apoptosis, vesicle trafficking, and cytoskeleton organization (5, 6).Three distinct classes of PI3Ks have now been identified on the basis of their in vitro substrate specificity, structure, and mode of regulation (7,8). The most studied are class I PI3Ks, which in vitro phosphorylate PtdIns, PtdIns(4)P, and PtdIns(4,5)P 2 , and display a preference for PtdIns(4,5)P 2 in vivo. Class I PI3Ks form a heterodimeric complex and are subdivided according to the adaptor protein associated with the catalytic subunit. Class IA PI3Ks consist of a 110-kDa catalytic subunit (p110 ␣, ␤, ␦) (9) and a 85-kDa adaptor protein (p85 ␣, ␤) (10) containing Src homology 2 (SH2) domains that link them to tyrosine kinase signaling. In contrast, c...

show abstract

“…As described previously (Breton et al, 1986), [35S]-sulfate incorporation was shown to be specific for the labeling of proteoglycans synthesized by pig smooth muscle cells.…”

Section: Measurement Of the Rate Of [35sl-labeledmentioning

confidence: 99%

Effect of endothelial‐cell‐conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

Berrou

Breton

Deudon

et al. 1988

Journal Cellular Physiology

Self Cite

View full text Add to dashboard Cite

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.

show abstract

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Cited by 32 publications

References 23 publications

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

Characterization of a G Protein-activated Phosphoinositide 3-Kinase in Vascular Smooth Muscle Cell Nuclei

Effect of endothelial‐cell‐conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

Contact Info

Product

Resources

About