Elizabeth A. Hawthorne

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.

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.

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 p27kip1 in vascular SMCs. p27kip1 messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27kip1, 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 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.

miR-221/222 Compensates for Skp2-Mediated p27 Degradation and Is a Primary Target of Cell Cycle Regulation by Prostacyclin and cAMP

p27kip1 (p27) is a cdk-inhibitory protein with an important role in the proliferation of many cell types. SCFSkp2 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 (PGI2), a potent cell cycle inhibitor acting through p27. PGI2 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 PGI2. PGI2 activates Gs to increase cAMP, and increasing intracellular cAMP phenocopies the effect of PGI2 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.

Matrix metalloproteinase-12 is an essential mediator of acute and chronic arterial stiffening.

Arterial stiffening is a hallmark of aging and risk factor for cardiovascular disease, yet its regulation is poorly understood. Here we use mouse modeling to show that matrix metalloproteinase-12 (MMP12), a potent elastase, is essential for acute and chronic arterial stiffening. MMP12 was induced in arterial smooth muscle cells (SMCs) after acute vascular injury. As determined by genome-wide analysis, the magnitude of its gene induction exceeded that of all other MMPs as well as those of the fibrillar collagens and lysyl oxidases, other common regulators of tissue stiffness. A preferential induction of SMC MMP12, without comparable effect on collagen abundance or structure, was also seen during chronic arterial stiffening with age. In both settings, deletion of MMP12 reduced elastin degradation and blocked arterial stiffening as assessed by atomic force microscopy and immunostaining for stiffness-regulated molecular markers. Isolated MMP12-null SMCs sense extracellular stiffness normally, indicating that MMP12 causes arterial stiffening by remodeling the SMC microenvironment rather than affecting the mechanoresponsiveness of the cells themselves. In human aortic samples, MMP12 levels strongly correlate with markers of SMC stiffness. We conclude that MMP12 causes arterial stiffening in mice and suggest that it functions similarly in humans.

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.