Devashish Kothapalli

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.

Phorbol Ester-induced G1 Phase Arrest Selectively Mediated by Protein Kinase Cδ-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 PKCδ 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 PKCδ, but not PKCα. 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 PKCδ but not PKCα. Studies using luciferase reporters also revealed an essential role for PKCδ in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCδ is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCδ 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.

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.

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.

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 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.

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.

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-β 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.

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.