miR-221/222 compensates for Skp2-mediated p27 degradation and is a primary target of cell cycle regulation by prostacyclin and cAMP.

p27(kip1) (p27) is a cdk-inhibitory protein with an important role in the proliferation of many cell types. SCF(Skp2) is the best studied regulator of p27 levels, but Skp2-mediated p27 degradation is not essential in vivo or in vitro. The molecular pathway that compensates for loss of Skp2-mediated p27 degradation has remained elusive. Here, we combine vascular injury in the mouse with genome-wide profiling to search for regulators of p27 during cell cycling in vivo. This approach, confirmed by RT-qPCR and mechanistic analysis in primary cells, identified miR-221/222 as a compensatory regulator of p27. The expression of miR221/222 is sensitive to proteasome inhibition with MG132 suggesting a link between p27 regulation by miRs and the proteasome. We then examined the roles of miR-221/222 and Skp2 in cell cycle inhibition by prostacyclin (PGI(2)), a potent cell cycle inhibitor acting through p27. PGI(2) inhibited both Skp2 and miR221/222 expression, but epistasis, ectopic expression, and time course experiments showed that miR-221/222, rather than Skp2, was the primary target of PGI(2). PGI(2) activates Gs to increase cAMP, and increasing intracellular cAMP phenocopies the effect of PGI(2) on p27, miR-221/222, and mitogenesis. We conclude that miR-221/222 compensates for loss of Skp2-mediated p27 degradation during cell cycling, contributes to proteasome-dependent G1 phase regulation of p27, and accounts for the anti-mitogenic effect of cAMP during growth inhibition.

Apolipoprotein E-mediated cell cycle arrest linked to p27 and the Cox2-dependent repression of miR221/222.

OBJECTIVE: In addition to its effects on cholesterol levels, apoE3 has lipid-independent effects that contribute to cardiovascular protection; one of these effects is the ability to inhibit cell cycling in VSMCs. The goal of this study was to identify and characterize cell cycle-regulatory mechanisms responsible for the anti-mitogenic effect of apoE. METHODS AND RESULTS: Primary VSMCs were stimulated with serum in the absence or presence of apoE3. apoE3 upregulated expression of the cdk inhibitor, p27(kip1), in primary VSMCs, and this effect required Cox2 and activation of PGI(2)-IP signaling. The microRNA family, miR221/222 has recently been identified as a post-translational regulator of p27, and apoE3 inhibited miR221/222 expression in a Cox2- and PGI(2)/IP-dependent manner. Moreover, reconstituted miR222 expression was sufficient to override the effects of apoE on p27 expression and S phase entry. The ability to repress expression of miR221/222 is shared by apoE3-containing HDL but is absent from apoA-1, LDL and apoE-depleted HDL. All three apoE isoforms regulate miR221/222, and the effect is independent of the C-terminal lipid-binding domain. miR221/222 levels are increased in the aortae of apoE3-null mice and reduced when apoE3 expression is reconstituted by adeno-associated virus infection. Thus, regulation of miR221/222 by apoE3 occurs in vivo as well as in vitro. CONCLUSIONS: ApoE inhibits VSMC proliferation by regulating p27 through miR221/222. Control of cell cycle-regulatory microRNAs adds a new dimension to the spectrum of cardiovascular protective effects afforded by apoE and apoE-HDL.

Cardiovascular protection by ApoE and ApoE-HDL linked to suppression of ECM gene expression and arterial stiffening.

Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein (apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.

Microsomal prostaglandin e2 synthase-1 modulates the response to vascular injury.

BACKGROUND: Microsomal (m) prostaglandin (PG) E2 synthase (S)-1 catalyzes the formation of PGE2 from PGH2, a cyclooxygenase product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than cyclooxygenase-2-selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation. The role of mPGES-1 in the response to vascular injury is unknown. METHODS AND RESULTS: Mice were subjected to wire injury of the femoral artery. Both neointimal area and vascular stenosis were significantly reduced 4 weeks after injury in mPGES-1 knockout mice compared with wild-type controls (65.6 ± 5.7 versus 37.7 ± 5.1 × 10³ pixel area and 70.5 ± 13.4% versus 47.7 ± 17.4%, respectively; P < 0.01). Induction of tenascin-C, a proproliferative and promigratory extracellular matrix protein, after injury was attenuated in the knockouts. Consistent with in vivo rediversion of PG biosynthesis, mPGES-1-deleted vascular smooth muscle cells generated less PGE2 but more PGI2 and expressed reduced tenascin-C compared with wild-type cells. Both suppression of PGE2 and augmentation of PGI2 attenuate tenascin-C expression and vascular smooth muscle cell proliferation and migration in vitro. CONCLUSIONS: Deletion of mPGES-1 in mice attenuates neointimal hyperplasia after vascular injury, in part by regulating tenascin-C expression. This raises for consideration the therapeutic potential of mPGES-1 inhibitors as adjuvant therapy for percutaneous coronary intervention.

Cell-type- and cell-cycle-specific anti-mitogenesis by cicaprost.

Stents eluting anti-proliferative drugs limit restenosis, but drugs commonly used to date are relatively non-specific cytostatic agents which inhibit proliferation of intimal endothelial cells as well as medial smooth muscle cells and may thereby contribute to the clinical complications associated with angioplasty. In an effort to identify a more specific anti-proliferative agent, we compared the effects of rapamycin to those of cicaprost, a mimetic of the naturally occurring anti-mitogen, PGI(2). Rapamycin and cicaprost were both strongly anti-mitogenic in vascular smooth muscle cells (VSMCs). But unlike rapamycin, cicaprost did not inhibit mitogenesis in aortic endothelial cells even when used at concentrations >10-fold higher than its ED(50) for VSMCs. Similarly, both rapamycin and cicaprost have been reported to regulate levels of the cdk inhibitor, p27(kip1). But rapamycin remained anti-mitogenic in p27(kip1)-null VSMCs whereas the anti-mitogenic effect of cicaprost was completely dependent on p27(kip1). We conclude that stable PGI(2) mimetics may be highly specific inhibitors of p27(kip1)-dependent VSMC proliferation after vascular injury.

Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.

BACKGROUND: A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle. RESULTS: We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo. CONCLUSIONS: Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.

Differential activation of ERK and Rac mediates the proliferative and anti-proliferative effects of hyaluronan and CD44.

Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.

Joint requirement for Rac and ERK activities underlies the mid-G1 phase induction of cyclin D1 and S phase entry in both epithelial and mesenchymal cells.

Cyclin D1 gene induction is a key event in G1 phase progression. Our previous studies indicated that signaling to cyclin D1 is cell type-dependent because the timing of cyclin D1 gene expression in MCF10A mammary epithelial cells and mesenchymal cells such as fibroblasts and vascular smooth muscle cells is very different, with epithelial cells first expressing cyclin D1 in early rather than mid-G1 phase. In this report, we induced a mesenchymal phenotype in MCF10A cells by long-term exposure to TGF-beta and used the control and transitioned cells to examine cell type specificity of the signaling pathways that regulate cyclin D1 gene expression. We show that early-G1 phase cyclin D1 gene expression in MCF10A cells is under the control of Rac, whereas mid-G1 phase cyclin D1 induction requires parallel signaling from Rac and ERK, both in the control and transitioned cells. This combined requirement for Rac and ERK signaling is associated with an increased requirement for intracellular tension, Rb phosphorylation, and S phase entry. A similar co-regulation of cyclin D1 mRNA by Rac and ERK is seen in primary mesenchymal cells. Overall, our results reveal two mechanistically distinct phases of Rac-dependent cyclin D1 expression and emphasize that the acquisition of Rac/ERK co-dependence is required for the mid-G1 phase induction of cyclin D1 associated with S phase entry.

Cell adhesion, cellular tension, and cell cycle control.

Cooperative signaling between growth factor receptor tyrosine kinases, integrins, and the actin cytoskeleton is required for activation of the G1-phase cyclin-dependent kinases and progression through G1-phase. Increasing evidence suggests that there is cell type specificity in these cooperative interactions and that the compliance of the underlying substratum can strongly affect adhesion-dependent signaling to the cell cycle. This chapter reviews our current methods for studying how cell type specificity and changes in substratum compliance can contribute to G1-phase cell cycle control. We also describe several of our current analytical procedures.

Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells.

High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031-1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27(kip1) in vascular SMCs. p27(kip1) messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27(kip1), was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury.

Antimitogenic effects of prostacyclin on the G1 phase cyclin-dependent kinases.

The prostanoid prostacyclin (PGI2) inhibits proliferation of cultured vascular SMCs by inhibiting cell cycle progression from G1 to S phase. Progression through G1 phase is regulated by the sequential activation of the G1 phase cyclin-dependent kinases (cdks). Recent studies have shown that PGI2-dependent activation of its receptor, IP, inhibits G1 phase progression by blocking the degradation of p27 and the activation of cyclin E-cdk2. High Density Lipoproteins (HDL) and its associated apolipoprotein, ApoE, also inhibit S phase entry of vascular SMCs, and the effects of HDL and ApoE are, at least in part, also mediated by the production of PGI2. The antimitogenic effects of hyaluronan may also be controlled by PGI2. This review summarizes the effects of PGI2 on the G1 phase cyclin-cdks and discusses the potential role of PGI2 as a common component of multiple extracellular signals that attenuate the proliferation of vascular SMCs.

Phorbol ester-induced G1 phase arrest selectively mediated by protein kinase Cdelta-dependent induction of p21.

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.

Antimitogenesis linked to regulation of Skp2 gene expression.

Prostacyclin has many effects in the vasculature; one of the less well understood is the ability to block cell cycle progression through G(1) phase. We previously reported that the prostacyclin mimetic, cicaprost, selectively inhibits cyclin E-cyclin-dependent kinase-2 (Cdk2), and now we show that it acts by regulating the expression of Skp2, the F-box protein that targets p27(Kip1) for ubiquitin-mediated proteolysis. First, we show that cicaprost prevents the late G(1) phase down-regulation of p27(Kip1) and that the inhibitory effect of cicaprost on cyclin E-Cdk2 activity and S phase entry is eliminated by deleting p27(Kip1). Levels of the closely related Cdk2 inhibitor, p21(Cip1), are unaffected by cicaprost. Moreover, we show that cicaprost blocks the induction of Skp2 mRNA and that ectopic expression of a Skp2 cDNA overrides the effect of cicaprost on p27(Kip1) levels and S phase entry. Our data show that inhibition of F-box protein gene expression can underlie the effect of a potent antimitogen.

Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation.

HDL and its associated apo, APOE, inhibit S-phase entry of murine aortic smooth muscle cells. We report here that the antimitogenic effect of APOE maps to the N-terminal receptor-binding domain, that APOE and its N-terminal domain inhibit activation of the cyclin A promoter, and that these effects involve both pocket protein-dependent and independent pathways. These antimitogenic effects closely resemble those seen in response to activation of the prostacyclin receptor IP. Indeed, we found that HDL and APOE suppress aortic smooth muscle cell cycle progression by stimulating Cox-2 expression, leading to prostacyclin synthesis and an IP-dependent inhibition of the cyclin A gene. Similar results were detected in human aortic smooth muscle cells and in vivo using mice overexpressing APOE. Our results identify the Cox-2 gene as a target of APOE signaling, link HDL and APOE to IP action, and describe a potential new basis for the cardioprotective effect of HDL and APOE.

Prostacylin receptor activation inhibits proliferation of aortic smooth muscle cells by regulating cAMP response element-binding protein- and pocket protein-dependent cyclin a gene expression.

The prostanoid prostacyclin (PGI2) inhibits aortic smooth muscle cell proliferation by blocking cell cycle progression from G1-to S-phase. However, the mechanism of this inhibition is poorly understood. We report here that the PGI2 mimetic, cicaprost, inhibits the induction of cyclin A and activation of the cyclin A promoter in primary and established rodent aortic smooth muscle cells. The inhibition of cyclin A gene expression is associated with a block in cyclin E-cdk2 activity and phosphorylation of both the retinoblastoma protein and p107. Inactivation of pocket proteins with human papilloma virus protein E7 partially, but not completely, restored cyclin A promoter activity in cicaprost-treated cells. Complementary studies showed that occupancy of the cAMP response element (CRE) is required for efficient activation of the cyclin A promoter in aortic smooth muscle cells, that the CRE is primarily occupied by the CRE-binding protein (CREB) and phospho-CREB, and that cicaprost blocks the binding of CREB and phospho-CREB to the cyclin A promoter CRE. Treatment with pertussis toxin reversed the inhibitory effects of cicaprost on CRE occupancy, cyclin E-cdk2 activity, and S phase entry, suggesting the involvement of Gi signaling in cicaprost action. We conclude that PGI2 inhibits proliferation of aortic smooth muscle cells by coordinately blocking CRE- and pocket protein-dependent cyclin A gene expression.