Elsevier

Atherosclerosis

Volume 133, Issue 1, August 1997, Pages 61-69
Atherosclerosis

G1 phase arrest of human smooth muscle cells by heparin, IL-4 and cAMP is linked to repression of cyclin D1 and cdk2

https://doi.org/10.1016/S0021-9150(97)00116-0Get rights and content

Abstract

Smooth muscle cell proliferation is a key event in the development of atherosclerosis. Inhibition of this proliferation may lead to better prevention and treatment of the disease. While a number of agents have been found to inhibit SMC proliferation, their mechanisms of action are not fully understood. We wanted to determine the effects of three physiologically relevant anti-mitogenic agents on two classes of proteins which have major roles in cellular proliferation, namely cyclins and cyclin-dependent kinases (cdks). Following stimulation with fetal calf serum (FCS), quiescent human umbilical artery smooth muscle cells (HUASMC) synthesised cyclin D1 mRNA and protein and cdk2 mRNA in the G1 phase, whereas cdc2 protein was expressed after the onset of the S phase. Heparin, a strong inhibitor of HUASMC proliferation, strongly down-modulated the levels of cyclin D1 mRNA and protein, cdk2 mRNA and cdc2 protein. Interleukin-4 (IL-4) or 8-bromo-adenosine 3′,5′- cyclic monophosphate (cAMP) also lowered the levels of these cell cycle regulatory proteins, although their effects were relatively weak, reflecting their only partial inhibition of HUASMC DNA synthesis. There was specificity in the cell cycle targets of the agents since none appeared to affect the levels of cdk4 protein.

Introduction

Atherosclerosis is a principal cause of heart disease, which in turn is the major cause of death in western countries. Although a key event in the pathogenesis of the disease is the migration and proliferation of smooth muscle cells (SMC) from the medial layer of the artery to the intima ([1]for review), the precise mechanisms which control arterial SMC proliferation have yet to be elucidated. Recently it has become clear that two classes of proteins play key roles in cell cycle progression in a wide range of cell types—namely the cyclins and their catalytic partners, cyclin dependent kinases (cdks) (see [2]for review). While the roles of cyclins and cdks have been investigated in many cell types, there are surprisingly few reports detailing their induction and regulation in SMC.

Phosphorylation of the retinoblastoma gene product (pRB) is an essential event required for proliferation of normal cells (see [3]for review). Current models suggest that hypophosphorylated pRB binds and thereby inhibits the transactivational activity of transcription factors such as E2F. Hyperphosphorylation of pRB results in the release of these transcription factors, allowing them to participate in the transcription of genes involved in cell proliferation (see [4]for review). Phosphorylation of pRB during the G1 phase of the cell cycle is performed, in part, by cyclin-dependent kinases (cdk) -4 or -6, depending upon the cell type 5, 6. Activation of cdk4/6 requires binding to a D-type cyclin 5, 6, and cyclin D levels, particularly those of D1, appear rate-limiting and essential for G1 phase progression 7, 8. Thus, binding to cyclins positively regulates cdk activity; however the activation of cdks is inhibited by the binding of `cdk inhibitors' (CKIs, see [9]for review).

While cdk4/6, in association with a cyclin D, is likely to play a key role in mid G1 progression, another cyclin-dependent kinase, cdk2 10, 11, 12, appears to be essential for late G1 to S phase transition 12, 13. While cdk2 complexes with cyclin E to mediate this transition [14], it can also be activated following binding to the S phase cyclin A 15, 16. Furthermore, cdk2 can also bind the G1 cyclins D2, D3 [17], although the physiological relevance of these interactions is yet to be determined. A key role for cdk2 in late G1 may be to maintain pRB in its hyperphosphorylated state following initial phosphorylation by cdk4 (see [3]for review). Another cyclin-dependent kinase required for successful cell cycle progression is cdc2 18, 19. In mammalian cells its activity appears essential for progression through mitosis 20, 21, and this activity is dependent on its association with cyclin B [22]. cdc2 may also have a role in S phase progression since it is able to be activated by cyclin A 23, 24, 25.

Recently there has been much interest in the mechanism of action of antiproliferative agents, with a variety of studies showing such agents may operate via effects on the cyclin/cdk/CKI system. The cell cycle protein targets of the agents are not predictable. Some data indicate that the effects of agents is cell specific, and some findings contradict the observations of others (see below). We have previously shown that cyclin D1 mRNA and protein levels are down-modulated by agents which elevate intracellular cAMP and cause G1 arrest in primary macrophages 26, 27, and similar data have been also reported using astrocytes [28]and fibroblasts [29]. Cyclin D1 may also be a target for other anti-mitogens since its levels have also been reported to be inhibited by TGFβ in HaCaT cells (a keratinocyte cell line) [30], and in intestinal epithelial cells [31]. Contrary to these findings, others have found that cAMP had no effect on cyclin D1 levels in a macrophage cell line [32], nor did TGFβ have any effect on levels of D-type cyclins in Mv1Lu mink lung epithelial cells [17]or 32D myeloid cells [33]. The effect of TGFβ on cdk4 levels also remains unclear since some reports show it reduces levels in HaCat 30, 33and Mv1Lu [17]cells, yet others report it has no such effect in HaCaT cells [34]. Another strategy by which antiproliferative agents prevent activation of cyclin/cdk complexes is through CKIs. In these instances levels of cyclins and cdks are often not affected. Agents known to work via these mechanisms include TGFβ 34, 35, 36, cAMP [37], rapamycin [38], vitamin D3 [39]and lovastatin [40]. The above findings highlight that while antiproliferative agents target cell cycle machinery the exact mechanisms differ depending upon the agent and the cell type.

In the present study we examined the effects of a number of physiologically relevant anti-mitogens on expression of cyclin D1, cdk4, cdk2 and cdc2 in human smooth muscle cells derived from umbilical artery. The agents employed were heparin, 8-bromo-adenosine 3′, 5′- cyclic monophosphate (8Br-cAMP) and interleukin 4 (IL-4). The former two have been shown to inhibit proliferation in a wide variety of SMC types 41, 42, 43, 44, 45, 46, 47, while IL-4 was recently described as anti-mitogenic for human umbilical artery smooth muscle cells (HUASMC) [43]. Importantly, this study into mechanisms contributing to the progression of atherosclerotic disease (i) examines the effect of these anti-mitogens on human arterial smooth muscle cells rather than SMC derived from non-human or non-arterial sources and (ii) uses normal cells since perturbations in cell cycle machinery are often present in immortalized cell lines.

Section snippets

Cell culture reagents

Reagents were purchased from the following sources: DMEM, trypsin, penicillin and streptomycin were purchased from ICN-Flow Laboratories, Sydney, Australia. Fetal calf serum (FCS) was from Commonwealth Serum Laboratories, Parkville, Australia and Falcon culture flasks and plates from Becton Dickenson, USA. Recombinant human IL-4 (1×107 U/mg) was obtained from Schering Plough, NJ. Heparin (from porcine mucous) was purchased from David Bull Laboratories, Victoria, Australia. Tritiated thymidine

Heparin, IL-4 and 8Br-cAMP inhibit DNA synthesis in HUASMC

We have previously reported that heparin is a strong inhibitor of DNA synthesis in HUASMC, while the inhibitory effect of IL-4 is only partial [43]. Raising intracelluar cAMP in SMC appears to inhibit proliferation to varying degrees depending upon the source of smooth muscle cells, since rabbit vascular SMC are strongly affected 46, 47, yet rat vascular SMC are only partially inhibited 44, 45. We firstly determined the effect of raising intracellular cAMP in our model, namely SMC derived from

Discussion.

Smooth muscle cell proliferation is a major event in the development of the atherosclerotic lesion [1]. Understanding the mechanisms which control this proliferation—and perhaps more particularly, understanding mechanisms by which SMC proliferation inhibitors act—may have important ramifications for management of heart disease. In the present study we investigated whether cyclins and cdks (regulators of cell cycle progression) were targets for the actions of agents which inhibit HUASMC

Acknowledgements

The authors are grateful to Drs Gino Vairo and Damian Myers for critical reading of the manuscript and Justin Schooneman for photography/imaging. This work was performed with the financial assistance of the National Health and Medical Research Council of Australia, and the National Heart Foundation of Australia.

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    Current address: AMRAD Pharmacia Biotech, 34 Wadhurst Drive, Boronia, Victoria 3155, Australia.

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    Current address: Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom.

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