Ca²⁺, Calmodulin, and Cyclins in Vascular Smooth Muscle Cell Cycle

Ca²⁺ is a major determinant of many biochemical processes in various cell types, from the beginning of new life and egg fertilization to the end of life and cell death.¹ In vascular smooth muscle (VSM), physiological resting levels of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the nanomolar range are necessary to maintain basal vascular tone.^2-4 VSM activation is associated with an increase in [Ca²⁺]_i in the micromolar range,^2-4 and large increases in VSM [Ca²⁺]_i have been identified in excessive vasoconstriction disorders such as hypertension and coronary vasospasm.^2,5 Activation of surface membrane receptors in VSM triggers increases in [Ca²⁺]_i due to Ca²⁺ release from the intracellular stores in the sarcoplasmic reticulum, and Ca²⁺ entry from the extracellular space through Ca²⁺ channels. Ca²⁺ then activates specific protein kinases and phospatases that are involved in VSM contraction and relaxation.^2,3,6 Ca²⁺ may also function as a second messenger to activate other signaling pathways such as cytosolic phospholipase A2α, phospholipase C, protein kinase C and phosphodiesterase.^6-8 An increase in [Ca²⁺]_i could also modulate plasma membrane channels and pumps such as Ca²⁺-activated K⁺ channels and the plasma membrane CaATPase (PMCA).^9,10 Additionally, Ca²⁺ may affect sarcoplasmic reticulum channels and pumps such as the inositol 1,4,5-trisphosphate (IP₃) receptor, the ryanodine-sensitive receptor and intracellular Ca²⁺ release channels, and the Ca²⁺ uptake pump (SERCA).^11,12

Calmodulin, a Ca²⁺ Sensor and Regulatory Protein

Ca²⁺ performs most of its functions by interacting with specific Ca²⁺ binding proteins, which serve as Ca²⁺ sensors and regulatory proteins. Calmodulin (CaM) is a critical Ca²⁺ sensor and regulatory protein in VSM. CaM is a ubiquitous small acidic protein of 16.7 kDa that is involved in virtually all Ca²⁺-dependent intracellular events.^2,3 The total intracellular concentration of CaM in the cell is significantly lower than the total concentration of its intracellular targets, making it a limiting factor in their regulation. CaM contains four EF-hand Ca²⁺ binding sites. The formation of Ca²⁺/CaM complex is necessary to activate myosin light chain (MLC) kinase, leading to MLC phosphorylation, actin-myosin interaction and VSM contraction. On the other hand, Ca²⁺/CaM-dependent protein phosphatase initiates MLC dephosphorylation and VSM relaxation. Also, Ca²⁺/CaM-dependent protein kinase II (CaMKII) is a ubiquitous mediator of Ca²⁺-linked signaling pathways that phosphorylates a wide range of substrates to coordinate and regulate Ca²⁺-mediated cellular functions.^13-15 For example, CaMKII phosphorylates and inactivates MLC kinase, a process that may be essential in the regulation of VSM contraction.¹⁶

Ca²⁺/CaM in VSM Cell Cycle and Growth

Vascular tissue injury in response to hypoxia, hyperlipidemia, oxidative stress and nicotine smoking is associated with increased production of growth factors/promoters such as hypoxia-inducible factor, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, angiotensin II, and endothelin-1.^17-22 The growth factors stimulate the expression and phosphorylation of ERK1/2 and JNK, and increase the DNA binding activity of activator protein-1 (AP-1) and nuclear factor-kB (NF-kB).^17,23 These nuclear events induce the phenotypic transformation of VSM into undifferentiated rapidly growing cells. VSM cell (VSMC) growth contributes to the pathogenesis of vascular hypertrophic disorders such as hypertension, atherosclerosis and vascular restenosis following angioplasty.^24-26

Studies have implicated Ca²⁺ as a regulator of mammalian cell cycle during early G₁ phase and near the G₁ to S phase transition.²⁷ However, the molecular mechanisms underlying the Ca²⁺-sensitivity of the G₁/S phase transition in VSM cell cycle have not been clearly elucidated. Also, while some studies have suggested a role for CaM in cell cycle and have shown that Ca²⁺/CaM is required for cell proliferation in both unicellular and multicellular eukaryotes,^27,28 the specific molecular targets of the Ca²⁺/CaM-dependent pathways are unclear.

Potential Ca²⁺/CaM-dependent targets include the serine/threonine phosphatase calcineurin and the family of multifunctional Ca²⁺/CaM-dependent protein kinases (CaMKs). In mammalian cells, both types of enzymes contribute to the regulation of cell cycle progression. However, the mechanism by which Ca²⁺/CaM and its downstream targets, particularly calcineurin and CaMKs, regulate key cell cycle-regulatory proteins, remains enigmatic. By understanding how Ca²⁺/CaM regulates cell cycle progression in normal mammalian cells, we would gain insight into how hormones control cell division, and how VSMCs coordinate Ca²⁺ and its downstream targets during the cell transformation into the rapidly growing proliferative phenotype.²⁸

Ca²⁺/CaM-Cyclin Interaction during VSM Cell Cycle

The passage of a cell through the cell cycle is controlled by proteins in the cytoplasm called cyclins. Recent studies have revealed the important role of cyclins in vascular and cardiac tissue injury, inflammation and wound repair.²⁹ Cyclins bind to and activate specific cyclin-dependent kinases (CDKs), which in turn phosphorylate a variety of protein substrates that control the cell cycle. While the levels of CDKs in the cell remain fairly stable, the levels of certain cyclins rise and fall with the specific stages of the cell cycle. Cyclin D and CDK4 mainly control the G₁ phase, cyclins A and E and CDK2 control the S-phase, while cyclins A and B and CDK1 control the mitotic phase. Growth factors stimulate the expression and tyrosine phosphorylation of ERK-1, which in turn promote the expression of cyclins and induce VSMC growth.²¹ On the other hand, a decrease in tyrosine phosphorylation of ERK1/2 reduces the expression of cyclins D1 and E, resulting in G₁ phase cell cycle arrest and inhibition of VSMC proliferation.³⁰

Studies have shown that depletion of Ca²⁺ pools in VSMC using the SERCA inhibitor thapsigargin causes inhibition of translocation of activated ERK1/2 to the nucleus, and prevents cyclin D1 expression, thus delaying the progression into the S-phase and the cell cycle.³¹ Also, forced gene expression of SERCA2a in a model of balloon injury of the rat carotid artery is associated with decreased cyclin D1 in VSMCs, cell cycle arrest at the G1 phase, and reduced VSMC proliferation and neointima formation.³² Although these studies suggest possible effects of intracellular Ca²⁺ on cyclin expression and the CDK activity, the specific molecular interactions and targets involved have not been clarified.

In this issue of Circulation Research Choi and colleagues describe a possible CaM binding site on cyclin E which could be involved in Ca²⁺-sensitive G₁/S transition in VSMCs.³³ They report that the kinase activity of cyclin E/CDK2 was responsive to physiological changes in Ca²⁺ concentration. Pharmacological inhibition of CaM using calmidazolium abrogated the Ca²⁺-sensitivity of cyclin E/CDK2, retarded VSMC proliferation and caused cell cycle arrest at G₁ phase. A highly conserved 22 aa N-terminal CaM-binding motif in cyclin E genes was essential in mediating the Ca²⁺-sensitive kinase activity of cyclin E/CDK2. Mutant cyclin E protein, lacking this CaM-binding motif, did not respond to alterations in Ca²⁺ concentration. These data clearly suggest that CaM-dependent cyclin E/CDK2 activity could mediate the Ca²⁺-sensitivity of the G₁/S transition in VSM cell cycle. These novel findings not only further elucidate the molecular mechanisms underlying VSMC growth, but may also help in the design of therapies that specifically aim at Ca²⁺/CaM-dependent cyclin E/CDK2 activity and can be used in the prevention/treatment of hypertrophic VSMC disorders. The study also raises interest in further understanding the role of Ca²⁺_, CaM and cyclins in VSMC proliferation and the factors that regulate the levels of the Ca²⁺/CaM complex and the cyclin/CDK activity.

Regulation of Ca²⁺/CaM in quiescent and growing VSMC

An important point relates to the level of Ca²⁺ that triggers the G₁/S transition. Because of its small molecular size, Ca²⁺ is expected to rapidly and homogeneously distribute inside the cell. However, some studies suggest localized Ca²⁺ gradients and sparks in the subplasmalemmal region and perinuclear sites.^4,34 Also, the regulation of Ca²⁺ in the nucleus, where important Ca²⁺-sensitive transcriptional processes reside, is a debated issue.³⁵ Cells may also show oscillations in [Ca²⁺]_i,⁴ and it is possible that the levels of the Ca²⁺/CaM complex may mirror those of the Ca²⁺ oscillations. In effect, the levels of Ca²⁺/CaM may play a role in the intracellular Ca²⁺ oscillatory behavior and in the regulation of the Ca²⁺ mobilization mechanisms. IP₃ receptors in the sarcoplasmic reticulum are tetrameric intracellular Ca²⁺ channels, the opening of which is regulated by both IP₃ and Ca²⁺. IP₃ receptors are biphasically regulated by cytosolic Ca²⁺, which binds to two distinct sites. IP₃ promotes channel opening by controlling whether Ca²⁺ binds to the stimulatory or inhibitory sites. Inhibition of IP₃ receptors by Ca²⁺ may require CaM. It is likely that one lobe of CaM tethers it to the IP₃ receptor, while the other lobe binds Ca²⁺ and then interact with a second site on the receptor to cause its inhibition.¹¹

Also, the CaATPase (commonly called the Ca²⁺ pump) is a fine-tuner of intracellular Ca²⁺. The plasma membrane CaATPase (PMCA) plays a role in Ca²⁺ extrusion. PMCA is a large enzyme, with 10 transmembrane domains and a C-terminal cytosolic tail that contains regulatory sites, including a CaM-binding domain.¹⁰ The sarcoplasmic reticulum CaATPase (SERCA) plays a role in Ca²⁺ reuptake. Reported post-translational modifications affecting SERCA pump activity involve N-glycosylation, glutathionylation and CaMKII-dependent phosphorylation.¹²

CaM may also be regulated by phosphorylation. CaM is phosphorylated both in vitro and in vivo by multiple serine/threonine and tyrosine protein kinases. Casein kinase II and MLC kinase are two serine/threonine kinases that have been implicated in this process. Also, within the tyrosine kinases involved in CaM phosphorylation are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor tyrosine kinases, such as the Src family kinases, Janus kinase 2, and p38Syk. CaM phosphorylation brings important consequences for the physiological cell function and the cell growth as the phosphoCaM species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems.³⁶

Regulation of cyclin/CDK activity

The activity of CDKs is controlled not only by Ca²⁺/CaM and cyclins, but also by CDK inhibitors (CKIs). Growth promotion is associated with upregulation of cyclins and CDKs and downregulation of CKIs, and the reverse occurs during growth arrest. The p16(INK4a). p21(Cip1/Waf1), p27(Kip1) and p57(Kip2) are known inhibitors of CDK.^23,37,38 Studies have shown that PPAR-α, a nuclear receptor that regulates lipid metabolism and inflammation, controls VSM cell cycle progression at the G₁/S transition by targeting the CKI p16(INK4a).³⁹ Also, downregulation of p27(kip1) and p57(kip2) in response to mitogenic stimulation plays a key role in the VSM cell cycle progression.³⁸ Additionally, the mitogenic effect of hyperlipemic sera and ox-LDL in VSMC may occur via inhibition of p21(Cip1) expression, and subsequent increase in DNA synthesis and cell proliferation.²² Furthermore, antioxidants not only downregulate cyclins D and E and CDKs 2 and 4, but also upregulate p21(Cip 1) and p27(Kip 1), leading to inhibition of CDK and arrest of cell cycle progression.²³ Nevertheless, it is not clear whether reduction in [Ca²⁺]_i and Ca²⁺/CaM would stimulate the CKIs.

Also, the potential role of newly discovered Ca²⁺ sensors and binding proteins in the regulation of cyclin/CDK activity and VSM cell cycle should be investigated. A novel Ca²⁺- binding protein called DREAM has been shown to interact with regulatory sequences of DNA, thereby acting as a Ca²⁺-dependent transcriptional regulator.⁴⁰ Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence. The transcriptional repressor DRE-antagonist modulator (DREAM) specifically binds to the DRE. DREAM contains four Ca²⁺- binding domains of the EF-hand type. Upon stimulation by Ca²⁺, DREAM’s ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca²⁺. Also, S100B, a dimeric EF-hand Ca²⁺-binding protein, interacts with the cell growth suppressor p53 and controls its transcriptional activity.⁴¹

Finally, the effect of other cations on the VSM cell cycle remains to be clarified. For instance, Mg²⁺ is known to affect Ca²⁺ entry into VSM and to counteract the effects of Ca²⁺ on VSM contraction. However, studies have shown VSM cell cycle activation and growth regulation by Mg²⁺ via ERK1/2-dependent, p38 MAP kinase-independent pathways.⁴²

Thus Ca²⁺ remains to be a master regulator of VSM contraction and growth. Several studies have clarified the role of CaM as a major Ca²⁺ sensor that regulates the activity of VSM channels, pumps and contractile proteins. New careful studies are now beginning to shed light on the intricate interaction between Ca²⁺, CaM, and cyclins in the regulation of VSM growth and proliferation.

Agonist (A)-receptor (R) interaction increases the hydrolysis of phosphatidylinositol 4,5- bisphosphate (PIP₂) and IP₃ production in VSM. IP₃ stimulates Ca²⁺ release from the sarcoplasmic reticulum (SR). Also, extracellular Ca²⁺ enters VSMC through Ca²⁺ channels. Ca²⁺ binds calmodulin (CaM), which could activate myosin light chain (MLC) kinase and initiate contraction, or regulate the activity of K⁺ channels, Ca²⁺ release channels, Ca²⁺ pumps, and CaM kinase II. During vascular injury, VSMC transforms into undifferentiated phenotype and enters a cell cycle which consists of G₁, growth and preparation of the chromosomes for replication; S, synthesis of DNA; G₂, preparation for mitosis; and M, mitosis. Ca²⁺/CaM increases the activity of cyclin E/CDK2 and stimulates G₁/S transition, and thereby promotes VSM growth and proliferation.