cAMP-mediated Induction of Cyclin E Sensitizes Growth-arrested Adipose Stem Cells to DNA Damage–induced Apoptosis

Abstract

The differentiation capacity of mesenchymal stem cells has been extensively studied, but little is known on cell cycle-related events in the proliferation and differentiation phases of these cells. Here, we demonstrate that exposure to cAMP-increasing agents inhibits proliferation of adipose stem cells (ASCs). This antiproliferative effect is associated with both reduced cdk2 activity and pRB phosphorylation. Concomitantly, however, the level of cyclin E markedly increases upon cAMP induction, indicating that cyclin E may have cdk2-independent functions in these cells besides its role as a cdk2 activator. Indeed, we found indications of a cdk2-independent role of cyclin E in DNA damage-induced apoptosis. 8-CPT-cAMP sensitizes ASCs to ␥-irradiation-induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated upregulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress-induced apoptosis in mesenchymal stem cells. INTRODUCTIONHuman mesenchymal stem cells (MSCs) have been shown to differentiate not only into mesenchymal lineages (such as osteogenic, chondrogenic, adipogenic, and myogenic), but also into neurogenic, angiogenic, and hepatic lineages (Zuk et al., 2002;Baksh et al., 2004;Boquest et al., 2006a;Schaffler and Buchler, 2007). The pluripotent nature of MSCs makes them potentially ideal candidates for tissue engineering, and they have already demonstrated some efficacy in cell therapy and regenerative medicine Kassem, 2004;Leo and Grande, 2006;Mimeault and Batra, 2006). Adipose tissue contains a supportive stroma that can be easily isolated and provides a rich source of MSCs (Zuk et al., 2002;Boquest et al., 2006a). Thus, adipose tissue represents a valuable source of multilineage stem cells. It is therefore important to understand the biology of adipose stem cells (ASCs).Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a number of different cellular processes, including metabolism, growth, differentiation, and gene regulation (Krebs and Beavo, 1979;McKnight, 1991;Vossler et al., 1997;Daniel et al., 1998;Kim et al., 2005). Most effects of cAMP are mediated through activation of protein kinase A (PKA) or the Epac (exchange protein directly activated by cAMP) family of exchange proteins (Walsh et al., 1968;Bos, 2003). Elevation of intracellular cAMP has both proliferative and antiproliferative effects depending on the cell type. For instance, cAMP stimulates the proliferation of thyroid cells, neurons, and Swiss 3T3 cells, while inhibiting the proliferation of lymphocytes, fibroblasts, and adipocytes (Rozengurt et al., 1981;Blomhoff et ...

cAMP induces autophagy via a novel pathway involving ERK, cyclin E and Beclin 1

“…8A) or knocking down MEK or cyclin E by siRNAs (Fig. 8B) blocked the PGE 2 -mediated perinuclear recruitment of Beclin 1, indicating that cAMP-induced recruitment is dependent on both the MEK-ERK pathway and cyclin E. Having previously demonstrated that induction of cyclin E by cAMP in ASCs depends on activation of ERK, 16 we have been able to establish the sequence of events from elevation of intracellular cAMP levels to induction of autophagy (Fig. 9).…”

“…5B and C), which is in line with our previous results on cAMP-signaling in ASCs. 16 As a control of equal loading, the lysates were also analyzed for expression of total ERK and/or actin.…”

“…6,9,10 The two former pathways have been shown to inhibit autophagy, 11,12 whereas activation of ERK can both stimulate or inhibit autophagy, depending on the cell type. 9,[13][14][15] Interestingly, activation of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway is known to either inhibit or activate ERK in a cell-type specific manner, [16][17][18][19][20] but in these studies the changes in ERK-activation have not been related to autophagy. In fact, only inhibition of autophagy has been observed upon activation of the cAMP/PKA pathway in the cell types studied so far.…”

“…19,[28][29][30] In a recent study, we showed that cAMP-signaling in adipocytederived mesenchymal stem cells (ASCs) resulted in activation of ERK, which in turn directed cyclin E to render these cells sensitive to DNA damage-induced apoptosis. 16 Cyclin E is generally considered to have a key function in driving the G 1 /S transition marker of autophagosome formation. 36 The conversion of LC3-I to LC3-II can be monitored by immunofluorescence analysis, as LC3 staining changes from a diffuse to punctate appearance when localized to autophagosomes, or by observing a shift in molecular weight upon protein gel blot analysis.…”

“…Enhancement of autophagic flux was also seen by treatment of cells with the of the cell cycle and in the initiation of DNA replication, [31][32][33] but in our study we demonstrated that cyclin E was elevated concomitantly with the cells being arrested in G 1 . 16 This led us to suggest that cAMP promotes a cell cycle-independent role of cyclin E in mesenchymal stem cells.…”