Regulation of the cell cycle is achieved through a complex and ordered sequence of events controlled by Cdk’s, the activation of which depends on their association with protein subunits and the cyclins, and on regulatory phosphorylation (12
). The activation of Cdk’s is negatively regulated by several CKIs. The four major mammalian CKIs fall into two classes. p21waf
are related proteins with a preference for Cdk2- and Cdk4-cyclin complexes, whereas p16INK4
are closely related CKIs that are specific for Cdk4- and Cdk6-cyclin complexes (12
). In the present study, we demonstrate that dm-LDL treatment leads to an accumulation of cells in G1 by increasing the level of p21waf
. Although a sustained increase in levels of p21waf
resulting from different stimuli could be mediated by various mechanisms (decreased protein degradation, increased mRNA translation rate, or increased mRNA half-life), a major mode of regulation is transcriptional (12
). Accordingly, our run-off experiments strongly support a role for transcription in regulating p21waf
expression upon dm-LDL stimulation.
was originally identified as a Cdk2 inhibitor whose activity, but not total protein amount, increased throughout the cell cycle (20
). This seemed to be associated with the release from intracellular compartments of p27 kip1
), and a similar mechanism might account for the stable expression of p27kip1
observed in our experiments.
The members of the STAT family undergo phosphorylation, dimerization, and nuclear translocation to activate target genes (9
). Among these, cell cycle–related genes are known to be transcriptionally regulated by STAT5 (48
). Moreover, it has been reported that during megakaryocyte differentiation, STAT5 regulates p21waf
). In the present study, we found that dm-LDL, but not n-LDL or ox-LDL, triggers STAT5 activation. Moreover, in nuclear extracts from dm-LDL–treated cells, the formation of a STAT5-containing p21SIE2-binding complex suggests that dm-LDL promotes p21waf
transcription through STAT5 activation. The molecular mechanisms underlying the activation of the STAT5 pathway by dm-LDL remain to be defined. In cytokine- or growth factor–mediated STAT activation, a ligand-dependent phosphorylation of the receptor creates a docking site for the Src homology 2 domain of STAT, thus recruiting STAT into the receptor complex (9
). It is known that modified LDL interacts with target cells through different receptors, such as the scavenger receptors (38
) and/or the receptor for the advanced glycated end products (39
). Indeed, our experiments showing that n-LDL was unable to compete with dm-LDL, to activate STAT5, or to induce ROS production suggest that n-LDL and dm-LDL bind to different receptors. Although we were unable to detect an immunoprecipitable complex between STAT5 and scavenger receptors for the advanced glycated end products (data not shown), it is possible that their association occurs either indirectly or through a low-affinity interaction. However, regardless of the mechanisms and of the receptor engaged by dm-LDL to elicit STAT5 activation, the findings that ectopic expression of ΔSTAT5B was able to alter dm-LDL–mediated upregulation of p21waf
expression, to rescue Cdk2 activity, and to prevent accumulation of cells in G1 strongly suggests a role for STAT5B in regulating this effect on endothelial cells.
Qualitative changes of LDL, such as glycation and/or oxidation, may account for the increased atherogenic risk in diabetes (2
). Indeed, dm-LDL did not show a significant degree of lipid peroxidation, but was characterized by a decreased cholesterol/apoB ratio and by a density profile showing a subfraction distribution corresponding to the LDL subclass defined as pattern B (49
). It is commonly accepted that, because of their susceptibility to glycation and/or oxidation, these small, dense particles are responsible for the increased atherogenic risk in diabetes (49
). Since dm-LDL did not bind to the canonical LDL receptor, did not activate a STAT3-mediated pathway (as shown by the inability of anti-STAT3 antiserum to modify the mobility shift of the p21SIE2-binding complex), and was recovered from patients in poor metabolic control, it is reasonable to assume that glycation, rather than oxidation, represents the qualitative change in dm-LDL that accounts for our results.
In conditions such as atherosclerosis, intimal angiogenesis occurs as part of the adaptive changes known as vasculature remodeling (50
). Recent clinical studies suggest that risk factors for coronary artery disease may modify an individual’s capacity for angiogenesis and vascular remodeling. Specifically, hypercholesterolemia and diabetes have been shown to be associated with a significant impairment in adaptive vascular growth of both capillary-like tube vessels and collateral vessels (51
). The observation that, through a STAT5B/p21waf
-mediated pathway, dm-LDL can affect the ability of endothelial cells to progress in the cell cycle adds further insight into the molecular mechanisms involved in the impaired vasculature remodeling in diabetes. Moreover, the evidence that activated STAT5 and p21waf
were highly expressed in endothelial cells lining both the luminal side of the plaque and/or the intimal neovessels supports the possibility that a similar mechanism may be operative in vivo.
In conclusion, the results presented here demonstrate that the natural plasma constituent LDL, from type 2 diabetic patients, can maintain endothelial cells in a quiescent state in G1 through STAT5B-mediated p21waf
expression. Moreover, the presence of a positive immunoreactivity for activated STAT5 and p21waf
in intraplaque neovessels supports the possibility that induction of STAT5-dependent genes may exert substantial atherogenic effects on the vessel wall, and specifically, may account for the deranged adaptive vascular growth observed in this pathological condition. Finally, the recent observation that NF-κB and STAT5 regulate the expression of the same gene in T cells (54
) raises the possibility that these transcriptional factors may also exert concerted effects on atherogenesis-related genes. However, further studies are required to elucidate the in vivo role of the STAT5 regulatory system.