Diabetic LDL inhibits cell-cycle progression via STAT5B and p21waf
Maria Felice Brizzi, … , Gianfranco Pagano, Luigi Pegoraro
Maria Felice Brizzi, … , Gianfranco Pagano, Luigi Pegoraro
Published January 1, 2002
Citation Information: J Clin Invest. 2002;109(1):111-119. https://doi.org/10.1172/JCI13617.
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Article

Diabetic LDL inhibits cell-cycle progression via STAT5B and p21waf

Abstract

Modified LDL is a major cause of injury to the endothelium in diabetes. In the present study, we analyzed the effects on endothelial cells of LDL recovered from type 2 diabetic patients (dm-LDL) or from nondiabetic subjects (n-LDL). Treatment of human umbilical vein endothelial cells with dm-LDL, but not n-LDL, led to the accumulation of cells in G1. To dissect the molecular mechanisms of this effect, we analyzed the expression and function of the cyclin-dependent kinase inhibitor p21waf, a cell cycle regulator known to be a target of the signal transducers and activators of transcription (STATs). dm-LDL led to transient STAT5 phosphorylation and the formation of a STAT5-containing complex and activated p21waf expression at the transcriptional level. Expression of the dominant-negative form of STAT5B, but not of STAT5A, significantly decreased both p21waf expression and the fraction of cells in G1. Finally, immunofluorescence analysis demonstrated that activated STAT5 is expressed in newly formed intraplaque vessels and in endothelial cells lining the luminal side of the plaque. Similarly, p21waf immunoreactivity was found in the neointimal vasculature. Our results suggest a role of STAT5B as a regulator of gene expression in diabetes-associated vascular disease.

Authors

Maria Felice Brizzi, Patrizia Dentelli, Marzia Pavan, Arturo Rosso, Roberto Gambino, Maria Grazia De Cesaris, Giovanni Garbarino, Giovanni Camussi, Gianfranco Pagano, Luigi Pegoraro

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Effect of the ΔSTAT5s on p21waf expression, p21SIE2-binding activity, an...
Effect of the ΔSTAT5s on p21waf expression, p21SIE2-binding activity, and Cdk2 kinase activity. (a) Effect of dominant-negative expression on endogenous STAT5. Cell extracts from ECV304 cells transfected with Neo vector (NEO), ΔSTAT5A (Δ5A), or ΔSTAT5B (Δ5B) cDNAs were stimulated for 10 minutes with IL-3 (10 ng/ml). Cell extracts were subjected to SDS-PAGE and processed as above. The filters were immunoblotted with anti–phospho-STAT5 and reprobed with an anti-STAT5 antiserum. Similar results were obtained in three different experiments. (b) Effect of ΔSTAT5 constructs on p21waf expression. Cells transfected with the different constructs were unstimulated (–) or stimulated for 18 hours (+) with n-LDL or dm-LDL. Cell extracts were processed, and the filters were immunoblotted with an anti-p21waf antiserum. (c) DNA-protein complex formation. Nuclear extracts from cells expressing ΔSTAT5A or ΔSTAT5B were stimulated with dm-LDL, processed, and resolved. The p21SIE2 complex is indicated. (d) Effect of p21waf expression on Cdk2-associated histone H1 kinase activity. Cells expressing Neo, ΔSTAT5A, or ΔSTAT5B cDNAs were stimulated with dm-LDL for 18 hours. Cell lysates were immunoprecipitated with an anti-Cdk2 antiserum and divided into two aliquots. One aliquot was subjected to an in vitro kinase assay (upper panel), fractionated by SDS-PAGE, and subjected to autoradiography. The second aliquot was subjected to SDS-PAGE and transferred to a nitrocellulose filter, and the filter was immunoblotted with an anti-Cdk2 antibody. Three different experiments were performed with similar results. IP, immunoprecipitation.