Open Access

The possible role of ribosomal protein S6 kinase 4 in the senescence of endothelial progenitor cells in diabetes mellitus

Contributed equally
Cardiovascular Diabetology201211:12

https://doi.org/10.1186/1475-2840-11-12

Received: 12 January 2012

Accepted: 2 February 2012

Published: 2 February 2012

Abstract

Background

The decrease and dysfunction of endothelial progenitor cells (EPCs) has been assumed as an important cause/consequence of diabetes mellitus (DM) and its complications, in which the senescence of EPCs induced by hyperglycemia may play an immensurable role. However, the mechanisms of EPCs senescence has not been fully investigated. Recently, ribosomal protein S6 kinase 4 (RSK4), a member of serine/threomine (Ser/Thr) kinase family and p53-related gene, is reported to regulate the replicative and stress-induced senescence of different cells.

Presentation of the hypothesis

These above lead to consideration of an evidence-based hypothesis that RSK4 may serve as a mediator of EPCs senescence in DM.

Testing the hypothesis

EPCs of healthy subjects and DM patients are isolated from peripheral blood and incubated with high glucose (HG). Then, the EPCs senescence would be detected by senescence associated β-galactosides (SA-β-gal) staining. Meanwhile, the RSK4 expression is assessed by RT-PCR and western blot. Moreover, overexpressing or RNA interfering of RSK4 in EPCs to investigate the relationship between RSK4 expression and the senescence of EPCs are necessary to substantiate this hypothesis. Also, studies on possible upstream and downstream factors of RSK4 would be explored to reveal the RSK4-mediated senescence pathway in EPCs.

Implications of the hypothesis

If proved, this hypothesis will provide another mediator of EPCs senescence, and may establish a novel pathogenesis for DM and further benefit to the management of DM.

Keywords

Diabetes mellitus Endothelial progenitor cell RSK4 Senescence

Background

EPCs are first reported in 1997 [1], which are derived from the bone marrow and could be mobilized to the peripheral circulation in response to stimuli. EPCs have been believed to be angioblasts and contribute to neovascularization, vascular maintenance and repair in adults, and EPCs dysfunction may enhance the risk for cardiovascular disease, DM and tumor [2, 3]. Emerging evidence has showed the count and function of EPCs are impaired in DM [46]. Moreover, diabetes could alter the subpopulation of EPCs by impairing the production in the bone marrow and decreasing the mobilization from the spleen [7]. Likewise, Jung C et. al found that DM patients had a smaller number of CD34-/CD133+ EPCs, but a larger proportion of apoptotic EPCs [8]. Besides, the reduction of EPCs may augment with an increased number of diseased coronary arteries, which may aggravate the DM and the complications [9]. And it is proved that increased EPCs number could promote the revascularization in asymptomatic type 2 diabetic patients [10, 11].

The mechanisms of EPCs dysfunction in DM

The mechanisms of the EPCs impairment are largely unknown. Reactive oxygen species (ROS) and nitric oxide (NO) are considered as regulators of EPCs [12]. Emerging evidence has found that hyperglycemia, as a type of ROS, could impair vascular endothelial function, and the severity of diabetes is reversely correlated with EPC number and function [13]. However, it is reported that EPCs could tolerate oxidative stress to some extent by upregulating superoxide dismutase (SOD), an enzyme that neutralizes superoxide anion (O2-) [14]. Similarly, Hamed S et.al found that EPCs from diabetic patients had higher SOD activity, but lower NO bioavailability than those from the healthy individuals. Nevertheless, when exposed to prolonged hyperglycemia in DM, the function of EPCs are adversely affected by excessive O2- generation [15], such as the reendothelialization capacity in vivo [16]. Furthermore, there also exit proofs that optimal glucose control could improve the number and function of EPCs [9, 17].

Recently, the senescence of EPCs has been assumed as an important cause/consequence of diabetes and its complications [18], the reasons of which lie in that hyperglycemia in vivo could product free radicals and generate oxidative stress, triggering cellular senescence in DM. However, the mechanisms remain largely unknown.

RSK4 and senescence

RSK4, ribosomal protein S6 kinase 4, is firstly found as an X-linked gene in patients with mental retardation and most abundantly expressed in brain and kidney [19]. As a member of Ser/Thr kinase family, RSK4 is widely participating in cell signaling pathway by regulating the proliferation and differentiation of cells [20, 21]. A large-scale RNAi screen in human cells identifies RSK4 as a new component of the p53 pathway, which could modulate the p53-dependent proliferation arrest on the p21cip1 promoter, either directly or indirectly [22]. Recently, it is reported that RSK4 could regulate replicative and stress-induced senescence [23], and the senescence could be bypassed when RSK4 is inhibited, of which is mediated by p21, but not of p16 or p38MAPKs [24].

RSK4 and diabetes

As a member of p90rsk family, RSK4 could modulate the synthesis of glucose. Insulin binding to its receptors results in interacting with growth factor receptor-bound protein 2 (Grb2). Grb2 is part of the cascades including RAS, RAF and MEK (MAP2K, Mitogen-activated protein kinase kinase) that leads to activation of mitogen-activated protein kinase (MAPK) and mitogenic responses [25], which includes the activation of glucose synthesis kinase (GSK), resulting in HG. As mentioned above, HG is the main diabetic feature and the cause of EPCs senescence in diabetes. Niehof et. al [26] found that RSK4 might provide a molecular rationale for late-stage complications of kidney and brain in Streptozotocin-induced diabetic rat with hepatic necrotic factor 4α (HNF4α) dysfunction. It is reported that HNF4α could regulate epithelial differentiation and overexpressed HNF4α could cause activation of p21 expression, a senescence mediator, thus inhibiting the cell proliferation [27]. Taken together, it is speculated that RSK4 may mediate EPCs senescence via p21 pathway in DM.

Presentation of the hypothesis

We assumed that RSK4 might serve as a mediator of EPCs senescence in DM. Hyperglycemia appears to be the most important cause of enhanced EPCs senescence. There are two main pathways involved in senescence: p19/p53 and p16/Rb [28]. p53 or p16 activates p21, which, in turn, can activate retinoblastoma protein (Rb) to shut down the transcription factor (E2F) target genes, thus inducing cell growth arrest and senescence [29]. Rosso et.al [30] reported that when cultured under HG, as a kind of ROS, normal EPCs underwent senescent-like growth arrest via the classical p53-dependent senescence pathway. Another study found that p16, together with telomerase, might co-modulate EPCs senescence. Besides, the activation of p38 MAPK pathway also involved in HG-induced EPCs senescence [31]. However, Chen et al. [32] reported that HG enhanced EPC senescence and impaired the migration and tube formation of late EPCs, which were modulated by NO-related rather than oxidative stress-mediated mechanisms through PI3K/Akt/eNOS signaling pathway. Another study [33] also showed PI3K/Akt/eNOS signaling cascade were suppressed in oxidized-LDL and HG treated EPCs, thus leading to the reduced number and the impaired functions of EPC in diabetic patients.

In addition, insulin resistance (IR) may also be a potential factor of EPCs senescence in diabetes. IR could lead to several biochemical alterations, including inflammation and oxidant stress, which leads to the dysfunction of EPCs via the following two pathways: PI3K-PDK1-Akt and RAS-MAPK-p38 pathway [3436]. However, it is not clear that whether this dysfunction of EPCs is senescence-related and what the senescence mediators are.

Based above, HG or IR could trigger signaling pathways, leading to the senescence of EPCs in DM. For now, PI3K-Akt-eNOS and p53-dependent pathway are considered to be linked to EPCs senescence [37].

Given that RSK4, a p53-related gene, participates in Ras-MEK-ERK pathway and could regulate senescence, we postulate that RSK4 might be a mediator in EPCs senescence in DM. If true, it will provide more information about the pathogenesis of diabetes and new therapeutic targets for diabetic patients. The possible signaling pathway of EPCs senescence is listed as Figure 1.
Figure 1

The possible signaling pathways of EPCs senescence. In diabetes, HG and/or IR might induce EPCs senesecne via the following pathways. HG and/or IR could inhibit the PI3K-Akt-eNOS pathway, resulting in the decrease of NO, which might induce the EPCs senescence. At the same time, HG and/or IR could be a kind of ROS and induce senescence through the classical p16 and p53 dependent senescence pathway, in which p38 is also invovled. Moreover, we conjecture that HG and/or IR could activate the insulin receptor mediated Ras-MEK-RSK4 pathway, resulting in on one hand the EPCs senescence mediated by RSK4 via p21 signaling pathway and a more production of glucose on the other hand. In additon, RSK4 could be a cadidate gene for HNF4α, which activates p21 and thus inhibit the cell proliferation in diabetes. HG: high glucose; IR: insulin resistance; ROS: reactive oxygen species; GSK: glucose synthesis kinase; HNF4α: hepatic necrotic factor 4α.

Testing the hypothesis

Our hypothesis demonstrates RSK4 protein might take a part in the senescence of EPCs. To testify the hypothesis, EPCs of healthy subjects and DM patients are isolated from peripheral blood and incubated with high glucose. Then, the EPCs senescence would be detected by SA-β-gal staining, and there might present an elevated number of SA-β-gal-positive EPCs. Meanwhile, the RSK4 expression is assessed by RT-PCR and western blot to find out whether it can be upregulated, which could provide an effective evidence for the hypothesis. Moreover, overexpressing or RNA interfering of RSK4 in EPCs to investigate the relationship between RSK4 expression and the senescence of EPCs are necessary to substantiate this hypothesis. Also, studies on possible upstream and downstream factors of RSK4 would be explored to reveal the RSK4-mediated senescence pathway in EPCs.

Implications of the hypothesis

As above, our new hypothesis might be another explanation to the EPCs senescence in DM. These findings may provide insight into a novel pathophysiological mechanism of DM and may offer new therapeutic opportunities in the future.

Notes

Abbreviations

Akt: 

PKB: protein kinase B

DM: 

Diabetes mellitus

E2F: 

Transcription factor

eNOS: 

Endothelial nitric oxide synthase

EPCs: 

Endothelial progenitor cells

ERK: 

Extracellular regulated protein kinases

Grb2: 

Growth factor receptor-bound protein 2

GSK: 

Glucose synthesis kinase

HG: 

High glucose

HNF4α: 

Hepatic necrotic factor 4α

IR: 

Insulin resistance

LDL: 

Low density lipoprotein

MAPK: 

Mitogen-activated protein kinase

MEK: 

MAP2K: mitogen-activated protein kinase kinase

NO: 

Nitric oxide

PDPK1: 

3-phosphoinositide-dependent protein kinase

PI3K: 

Phosphatidylinositol 3-kinase

Rb: 

Retinoblastoma protein

ROS: 

Reactive oxygen species

RSK4: 

Ribosomal protein S6 kinase 4

SA-β-gal: 

Senescence associated β-galactosides

Ser/Thr: 

Serine/threomine

SOD: 

Superoxide dismutase.

Declarations

Acknowledgements

The work is supported by the National Natural Science Foundation of China (2011 81001140).

Authors’ Affiliations

(1)
Department of Cardiology, Xijing Hospital, Fourth Military Medical University
(2)
State Key Laboratory of Cancer Biology and Department of Pathology, Xijing Hospital, Fourth Military Medical University

References

  1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997, 275 (5302): 964-967. 10.1126/science.275.5302.964.View ArticlePubMedGoogle Scholar
  2. Loomans CJ, De Koning EJ, Staal FJ, Rabelink TJ, Zonneveld AJ: Endothelial progenitor cell dysfunction in type 1 diabetes: another consequence of oxidative stress?. Antioxid Redox Signal. 2005, 7 (11-12): 1468-1475. 10.1089/ars.2005.7.1468.View ArticlePubMedGoogle Scholar
  3. Nolan DJ, Ciarrocchi A, Mellick AS, Jaggi JS, Bambino K, Gupta S, Heikamp E, McDevitt MR, Scheinberg DA, Benezra R, et al: Bone marrow-derived endothelial progenitor cells are a major determinant of nascent tumor neovascularization. Genes Dev. 2007, 21 (12): 1546-1558. 10.1101/gad.436307.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, et al: Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol. 2005, 45 (9): 1449-1457. 10.1016/j.jacc.2004.11.067.View ArticlePubMedGoogle Scholar
  5. Mohler ER, Shi Y, Moore J, Bantly A, Hamamdzic D, Yoder M, Rader DJ, Putt M, Zhang L, Parmacek M, et al: Diabetes reduces bone marrow and circulating porcine endothelial progenitor cells, an effect ameliorated by atorvastatin and independent of cholesterol. Cytometry A. 2009, 75 (1): 75-82.PubMed CentralView ArticlePubMedGoogle Scholar
  6. Georgescu A: Vascular dysfunction in diabetes: the endothelial progenitor cells as new therapeutic strategy. World J Diabetes. 2011, 2 (6): 92-97. 10.4239/wjd.v2.i6.92.PubMed CentralView ArticlePubMedGoogle Scholar
  7. Saito H, Yamamoto Y, Yamamoto H: Diabetes alters subsets of endothelial progenitor cells that reside in blood, bone marrow and spleen. Am J Physiol Cell Physiol. 2011.Google Scholar
  8. Jung C, Rafnsson A, Shemyakin A, Bohm F, Pernow J: Different subpopulations of endothelial progenitor cells and circulating apoptotic progenitor cells in patients with vascular disease and diabetes. Int J Cardiol. 2010, 143 (3): 368-372. 10.1016/j.ijcard.2009.03.075.View ArticlePubMedGoogle Scholar
  9. Bozdag-Turan I, Turan RG, Turan CH, Ludovicy S, Akin I, Kische S, Arsoy NS, Schneider H, Ortak J, Rehders T, et al: Relation between the frequency of CD34+ bone marrow derived circulating progenitor cells and the number of diseased coronary arteries in patients with myocardial ischemia and diabetes. Cardiovasc Diabetol. 2011, 10: 107-10.1186/1475-2840-10-107.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Kim HM, Kim KJ, Moon JH, Lee HJ, Chae MK, Chang HJ, Kang ES, Cha BS, Lee HC, Kim YJ, et al: Association between EPCs count and rate of coronary revascularization in asymptomatic type 2 diabetic patients. Acta Diabetol. 2011.Google Scholar
  11. Grapensparr L, Olerud J, Vasylovska S, Carlsson PO: The therapeutic role of endothelial progenitor cells in Type 1 diabetes mellitus. Regen Med. 2011, 6 (5): 599-605. 10.2217/rme.11.45.View ArticlePubMedGoogle Scholar
  12. Hamed S, Brenner B, Roguin A: Nitric oxide: a key factor behind the dysfunctionality of endothelial progenitor cells in diabetes mellitus type-2. Cardiovasc Res. 2011, 91 (1): 9-15. 10.1093/cvr/cvq412.View ArticlePubMedGoogle Scholar
  13. Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC: Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002, 106 (22): 2781-2786. 10.1161/01.CIR.0000039526.42991.93.View ArticlePubMedGoogle Scholar
  14. Dernbach E, Urbich C, Brandes RP, Hofmann WK, Zeiher AM, Dimmeler S: Antioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress. Blood. 2004, 104 (12): 3591-3597. 10.1182/blood-2003-12-4103.View ArticlePubMedGoogle Scholar
  15. Hamed S, Brenner B, Aharon A, Daoud D, Roguin A: Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus. Cardiovasc Diabetol. 2009, 8: 56-10.1186/1475-2840-8-56.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Sorrentino SA, Bahlmann FH, Besler C, Muller M, Schulz S, Kirchhoff N, Doerries C, Horvath T, Limbourg A, Limbourg F, et al: Oxidant stress impairs in vivo reendothelialization capacity of endothelial progenitor cells from patients with type 2 diabetes mellitus: restoration by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone. Circulation. 2007, 116 (2): 163-173. 10.1161/CIRCULATIONAHA.106.684381.View ArticlePubMedGoogle Scholar
  17. Palombo C, Kozakova M, Morizzo C, Gnesi L, Barsotti MC, Spontoni P, Massart F, Salvi P, Balbarini A, Saggese G, et al: Circulating endothelial progenitor cells and large artery structure and function in young subjects with uncomplicated Type 1 diabetes. Cardiovasc Diabetol. 2011, 10: 88-10.1186/1475-2840-10-88.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Testa R, Ceriello A: Pathogenetic loop between diabetes and cell senescence. Diabetes Care. 2007, 30 (11): 2974-2975. 10.2337/dc07-1534.View ArticlePubMedGoogle Scholar
  19. Yntema HG, van den Helm B, Kissing J, van Duijnhoven G, Poppelaars F, Chelly J, Moraine C, Fryns JP, Hamel BC, Heilbronner H, et al: A novel ribosomal S6-kinase (RSK4; RPS6KA6) is commonly deleted in patients with complex X-linked mental retardation. Genomics. 1999, 62 (3): 332-343. 10.1006/geno.1999.6004.View ArticlePubMedGoogle Scholar
  20. Dummler BA, Hauge C, Silber J, Yntema HG, Kruse LS, Kofoed B, Hemmings BA, Alessi DR, Frodin M: Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types. J Biol Chem. 2005, 280 (14): 13304-13314. 10.1074/jbc.M408194200.View ArticlePubMedGoogle Scholar
  21. Me LL, Vidal F, Gallardo D, Diaz-Fuertes M, Rojo F, Cuatrecasas M, Lopez-Vicente L, Kondoh H, Blanco C, Carnero A, et al: New p53 related genes in human tumors: significant downregulation in colon and lung carcinomas. Oncol Rep. 2006, 16 (3): 603-608.Google Scholar
  22. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, Kerkhoven RM, Madiredjo M, Nijkamp W, Weigelt B, et al: A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature. 2004, 428 (6981): 431-437. 10.1038/nature02371.View ArticlePubMedGoogle Scholar
  23. Lopez-Vicente L, Armengol G, Pons B, Coch L, Argelaguet E, Lleonart M, Hernandez-Losa J, de Torres I, Ramon y, Cajal S: Regulation of replicative and stress-induced senescence by RSK4, which is down-regulated in human tumors. Clin Cancer Res. 2009, 15 (14): 4546-4553. 10.1158/1078-0432.CCR-08-3159.View ArticlePubMedGoogle Scholar
  24. Lopez-Vicente L, Pons B, Coch L, Teixido C, Hernandez-Losa J, Armengol G, Ramon YCS: RSK4 Inhibition Results in Bypass of Stress-Induced and Oncogene-Induced Senescence. Carcinogenesis. 2011, 32 (4): 470-476. 10.1093/carcin/bgr003.View ArticlePubMedGoogle Scholar
  25. Carriere A, Ray H, Blenis J, Roux PP: The RSK factors of activating the Ras/MAPK signaling cascade. Front Biosci. 2008, 13: 4258-4275.View ArticlePubMedGoogle Scholar
  26. Niehof M, Borlak J: RSK4 and PAK5 are novel candidate genes in diabetic rat kidney and brain. Mol Pharmacol. 2005, 67 (3): 604-611.View ArticlePubMedGoogle Scholar
  27. Chiba H, Itoh T, Satohisa S, Sakai N, Noguchi H, Osanai M, Kojima T, Sawada N: Activation of p21CIP1/WAF1 gene expression and inhibition of cell proliferation by overexpression of hepatocyte nuclear factor-4alpha. Exp Cell Res. 2005, 302 (1): 11-21. 10.1016/j.yexcr.2004.08.014.View ArticlePubMedGoogle Scholar
  28. Maity A, Koumenis C: HIF and MIF-a nifty way to delay senescence?. Genes Dev. 2006, 20 (24): 3337-3341. 10.1101/gad.1506906.View ArticlePubMedGoogle Scholar
  29. Itahana K, Campisi J, Dimri GP: Mechanisms of cellular senescence in human and mouse cells. Biogerontology. 2004, 5 (1): 1-10.View ArticlePubMedGoogle Scholar
  30. Rosso A, Balsamo A, Gambino R, Dentelli P, Falcioni R, Cassader M, Pegoraro L, Pagano G, Brizzi MF: p53 Mediates the accelerated onset of senescence of endothelial progenitor cells in diabetes. J Biol Chem. 2006, 281 (7): 4339-4347. 10.1074/jbc.M509293200.View ArticlePubMedGoogle Scholar
  31. Kuki S, Imanishi T, Kobayashi K, Matsuo Y, Obana M, Akasaka T: Hyperglycemia accelerated endothelial progenitor cell senescence via the activation of p38 mitogen-activated protein kinase. Circ J. 2006, 70 (8): 1076-1081. 10.1253/circj.70.1076.View ArticlePubMedGoogle Scholar
  32. Chen YH, Lin SJ, Lin FY, Wu TC, Tsao CR, Huang PH, Liu PL, Chen YL, Chen JW: High glucose impairs early and late endothelial progenitor cells by modifying nitric oxide-related but not oxidative stress-mediated mechanisms. Diabetes. 2007, 56 (6): 1559-1568. 10.2337/db06-1103.View ArticlePubMedGoogle Scholar
  33. Hamed S, Brenner B, Abassi Z, Aharon A, Daoud D, Roguin A: Hyperglycemia and oxidized-LDL exert a deleterious effect on endothelial progenitor cell migration in type 2 diabetes mellitus. Thromb Res. 2010, 126 (3): 166-174. 10.1016/j.thromres.2010.03.002.View ArticlePubMedGoogle Scholar
  34. Cubbon RM, Kahn MB, Wheatcroft SB: Effects of insulin resistance on endothelial progenitor cells and vascular repair. Clin Sci (Lond). 2009, 117 (5): 173-190. 10.1042/CS20080263.View ArticleGoogle Scholar
  35. Cubbon RM, Rajwani A, Wheatcroft SB: The impact of insulin resistance on endothelial function, progenitor cells and repair. Diab Vasc Dis Res. 2007, 4 (2): 103-111.View ArticlePubMedGoogle Scholar
  36. Desouza CV, Hamel FG, Bidasee K, O'Connell K: Role of inflammation and insulin resistance in endothelial progenitor cell dysfunction. Diabetes. 2011, 60 (4): 1286-1294. 10.2337/db10-0875.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Everaert BR, Van Craenenbroeck EM, Hoymans VY, Haine SE, Van Nassauw L, Conraads VM, Timmermans JP, Vrints CJ: Current perspective of pathophysiological and interventional effects on endothelial progenitor cell biology: focus on PI3K/AKT/eNOS pathway. Int J Cardiol. 2010, 144 (3): 350-366. 10.1016/j.ijcard.2010.04.018.View ArticlePubMedGoogle Scholar

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© Yin et al; BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.