News Physiol Sci 15: 41-45, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 1, 41-45,
February 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
Phospholipase C Isoforms, Cytoskeletal Organization, and Vascular Smooth Muscle Differentiation
Joanne S. Lymn and
Alun D. Hughes
J. S. Lymn and A. D. Hughes are in the Department of Clinical Pharmacology, National Heart and Lung Institute, Imperial College School of Medicine, QEQM Wing, St. Mary's Hospital, Paddington, London W2 1NY, England.
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Abstract
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The function of differentiated vascular smooth muscle cells (VSMC) in vivo is the regulation of contractility. Following injury or disease, however, VSMC lose their contractile function and take on a synthetic, proliferative phenotype. This dedifferentiation is generally accompanied by a change in the expression profile of phospholipase C isoforms.
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Introduction
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Conventional inositol phospholipid metabolism involves the sequential phosphorylation of phosphatidylinositol (PI) to PI 4-phosphate (PIP) and then to PI 4,5-bisphosphate (PIP2). PIP2 is the in vivo substrate for the phospholipase C (PLC) family of enzymes. PLC hydrolyzes PIP2 to give inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). Both products of this reaction act as key second messengers in a number of signaling pathways, including those leading to vascular smooth muscle (VSM) growth and contraction.
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Regulation of PLC isoforms
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The phosphoinositide-specific superfamily of PLC enzymes currently contains 10 members, which can be classified into three families of PLC isoforms, termed ß, 
, and 
. All mammalian PLC isoforms contain four common domains [catalytic, comprising X and Y regions; C2; plekstrin homology (PH); and EF-hand], recognize PI, PIP, and PIP2, and carry out the calcium-dependent hydrolysis of these inositol lipids. However, these isoforms are differentially regulated (7). PLC-ß, of which there are currently four isoforms described (PLC-ß1 through -ß4), is regulated by heterotrimeric G proteins in response to agonist binding to serpentine receptors. PLC-ß1, -ß3, and -ß4 are regulated by binding of the 
-subunit of the pertussis toxin-insensitive Gq family of heterotrimeric G proteins, whereas PLC-ß2 is thought to be largely regulated by G protein ß-subunits. PLC-, comprising PLC-1 and -2, contains two types of src homology (SH) domains and is thought to be regulated by tyrosine phosphorylation following binding to either growth factor-activated receptor tyrosine kinases or to cytosolic tyrosine kinases of the src family. The mechanism of regulation of PLC-, probably the earliest isoform of PLC in evolutionary terms, remains largely obscure. PLC- does not have a long carboxy terminus nor does it contain SH domains. However, it has been demonstrated to bind very strongly to both PIP2 and IP3 and has recently been linked to both a novel GTP-binding protein Gh and to the actin cytoskeleton via the monomeric GTPase RhoA (Fig. 1
).
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FIGURE 1. Schematic representation of functional domains found in each of the 3 isoforms of phospholipase C (PLC), including core domains [plekstrin homology (PH), EF-hand, X, Y, and C2] common to all PLC isoforms and those specific for individual isoforms. -Subunits of the Gq family of heterotrimeric G proteins bind to a region called the G box present in long carboxy tail of PLC-ß isoforms. Heterotrimeric G protein ß-subunits interact with amino terminal portion of PLC-ß isoforms, possibly with part of the catalytic or PH domains. PLC- isoforms contain two src homology 2 (SH2) domains that interact with phosphotyrosine residues and an SH3 domain that can interact with proline-rich regions. Inositol 1,4,5-trisphosphate (IP3) binds strongly to PH domain of PLC- and in a negative feedback mechanism. PIP2, phosphatidylinositol 4,5-biphosphate.
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This differential regulation of PLC isoforms has long been regarded as the mechanism that allows very different cell stimulants to access a single biochemical event, namely the production of IP3 and DAG. A wider implication of this differential regulation, however, may be differential cellular function.
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Functional role of PLC- isoforms
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PLC- is generally stimulated by growth factors and, as such, is a prime candidate for the induction of cell growth and proliferation, although the absolute requirement of this enzyme for mitogenic signaling is species and cell-type dependent.
Recent work examining the differential expression of rat brain PLC isoforms in development and ageing demonstrated that PLC- activity was highest in fetal tissue and suggested that this isoform was therefore particularly involved in cell division and growth. Similarly, PLC- expression is significantly elevated in colorectal and human breast carcinomas compared with normal tissue and is also highly expressed in a number of colon carcinoma cell lines (8). Indeed, the new class of PI-PLC-1 inhibitors recently isolated from the sarcotestas of Ginkgo biloba have been shown to inhibit the growth of a number of human cancer cell lines more than normal human colon cell lines.
Not surprisingly, overexpression of wild-type PLC-1 has been shown to induce DNA synthesis and alter both the growth rate and the morphology of quiescent fibroblasts. What is surprising is the finding that lipase-deficient mutants of PLC-1 can also induce DNA synthesis in quiescent fibroblasts, although not to maximal levels. Coinjection of IP3 and DAG with the lipase-defective PLC-1 mutant restored maximal DNA synthesis; thus catalytic activity potentiated the mitogenic response under these circumstances. This suggests that regions other than the catalytic domain may be responsible, at least in part, for mitogenic signaling. Further studies in this area have demonstrated that microinjection of the complete SH domain of PLC-1 induces a 25-fold increase in DNA synthesis in quiescent fibroblasts, whereas microinjection of the PH domain had no effect. Fragments of the complete SH domain, including the two SH2 domains and the SH3 domain preceded by tyrosine phosphorylated residues at positions 771 and 783, each induced a partial response. These responses were additive on coinjection (12). Intriguingly, there is also increasing evidence from a number of tissues, including VSM, that tyrosine phosphorylation, although undoubtedly necessary, may not always be sufficient to induce full activation of PLC-. Indeed, tyrosine phosphorylation of PLC- may instead be critical for translocation of the enzyme from the cytosol to the actin cytoskeleton (15). Platelet-derived growth factor stimulation of fibroblasts results in the localization of tyrosine 783 phosphorylated PLC-1 at membrane ruffles and stress fibers, where it colocalized with actin filaments. Both this localization and the subsequent depolymerization of actin filaments can be prevented by injection of both an antibody to tyrosine 783 phosphorylated PLC-1 and by a PLC-1-2SH2 domain protein. Intriguingly, microinjection of a 13-amino acid phosphorylated peptide, containing tyrosine 783 alone, can also induce disassembly of actin filaments and membrane ruffling in fibroblasts, suggesting that catalytic activity is not necessary for actin rearrangement (Fig. 2). In accordance with these studies, the introduction of a mutant PLC-1 in which phenylalanine had been substituted for tyrosine at position 783 resulted in cells taking on a different morphology from those expressing wild-type PLC-1, exhibiting both much thicker actin filaments and reduced DNA synthesis (10).
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FIGURE 2. Mitogenic signaling through PLC-1 without production of IP3 and 1,2-diacylglycerol (DAG). Schematic depicts effects of complete SH domain, or fractions thereof, on actin cytoskeleton and DNA synthesis. PIP, phosphatidylinositol 4-phosphate.
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Clearly then, PLC- isoforms are implicated in mitogenic signaling, with a major role of these isoforms being the reorganization of the actin cytoskeleton, which can occur independently of the catalytic activity of this enzyme.
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Functional role of PLC- isoforms
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In contrast to PLC-, PLC- isoforms have not been implicated in cell growth and proliferation. Indeed, PLC- expression in colon carcinomas is decreased compared with normal tissue, and PLC- expression has not been detected in any of the human carcinoma cell lines examined (8). Similarly, PLC- expression was not detected in the large bowel neoplasm of methylazoxymethanol acetate-treated rats, although it was readily detected in both the nonneoplastic colonic mucosa of these rats and in control rats. In fact, the inverse correlation between PLC- expression and ornithine decarboxylase activity led to the conclusion that PLC- has little to do with PLC-mediated mitogenic signaling (14). Indeed, Rhee et al. (11) commented that PLC- exhibits limited expression in cultured cells, although it is routinely detected in tissues and is the predominant isoform of PLC in differentiated muscle cells (5).
Although understanding the mechanism of regulation of PLC- is in its infancy, there is enough accumulated information about this isoform for us to speculate on its possible cellular function(s). PLC-1 binds to both PIP2 and IP3 with high affinity through a three-dimensional binding pocket, which exists in native PLC-1 or the associated PH domain of this isoform. A similar site has not been detected in any of the other PLC isoforms studied, suggesting that PLC-1 binds PIP2 with a uniquely high affinity. The PH domain of PLC-1 has therefore been suggested to direct the host protein to membranes enriched with PIP2 and to be subject to product inhibition. Analysis of this binding affinity and the limited amount of PIP2 present in cell membranes has led to the suggestion that, under physiological conditions, a considerable fraction of PLC-1 may be bound to the cell membrane. PLC- is likely to be bound to PIP2 in either an inactive form or an inhibited form under resting conditions; otherwise, rapid substrate hydrolysis would occur. Interestingly, recent data, including that from this laboratory, have linked PLC- with the monomeric GTPase RhoA. Homma and Emori (4) isolated a novel Rho GTPase-activating protein that had PLC- stimulatory activity; similarly, we have shown that bovine aortic PLC-1 is associated with RhoA and that Clostridium botulinum exoenzyme C3 (exoenzyme C3), which ADP-ribosylates and inactivates RhoA, leads to an increase in PLC- activity (3). This raises the possibility that RhoA may act as an inhibitory modulator of PLC- activity (Fig. 3) and potentially links PLC- with the process of cytoskeletal organization.
Rho family proteins (e.g., Rho, Rac, and Cdc42) have clearly been demonstrated to play a major role in the organization of the actin cytoskeleton of a number of cell types, with Rho itself promoting the formation of actin stress fibers and focal adhesion complexes. These monomeric GTPases have also been linked to the stimulation of PIP2 synthesis; similarly, PIP2 has itself been linked to a number of actin-binding proteins, including both profilin and gelsolin.
Conversely, perhaps both Rho guanosine 5'-O-(3-thiotriphosphate) (GTPS) and constitutively active Rho have been shown to enhance the calcium sensitivity of smooth muscle contraction, and both exoenzyme C3 and Staphylococcus aureus epidermal differentiation inhibitor (EDIN), which inhibit Rho activity, have been shown to inhibit agonist-induced increases in calcium sensitivity. This suggests a possible role for RhoA in promoting contractility in smooth muscle, possibly as a result of regulation of myosin phosphatase activity. Although direct evidence implicating PLC- in these processes is lacking at present, it is tempting to suggest that this isoform is involved in the modulation of the actin cytoskeleton through its close associations with both PIP2 and RhoA. Furthermore, this opens the possibility that this isoform of PLC may play a role in modulating the contractile ability of cells. Indeed, recent data have suggested that PLC-, through stimulation of Gh, is an effector for both the contractile agonist noradrenaline and the hormone oxytocin, which is critical for the induction of uterine contraction at term (1, 9).
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Differentiated VSM
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The primary function of VSM in mature animals is contraction. Indeed, fully differentiated smooth muscle cells in mature blood vessels proliferate at extremely low rates. Differentiated smooth muscle cells are highly specialized and express a variety of characteristic contractile and cytoskeletal proteins, which are involved in the regulation of contraction. Culture of VSM cells results in phenotypic modulation, with the cells undergoing morphological changes and proliferating at greatly enhanced rates. These "synthetic" cells show alterations in contractile and cytoskeletal proteins (e.g., decreased expression of -actin and myosin heavy chain) and rapidly become noncontractile. A comparable process is believed to occur in intact arteries after injury, in which smooth muscle cells dedifferentiate before migration into (and proliferation within) the intima. At least in some circumstances, this phenotypic modulation may be reversible in vivo; however, the phenotypic modulation that VSM cells undergo in culture is not reversible by any current technique. Some differentiation markers, including expression of contractile proteins and functional voltage-gated calcium channels, can be recovered to some degree by culture on Matrigel or by deep growth arrest. Nevertheless, these cells remain noncontractile. Intriguingly, intact vessels maintained in organ culture also show a progressive decrease in contractility with time in culture. This process is accelerated by the presence of fetal calf serum and is accompanied by an increase in DNA synthesis and protein content but is not associated with obvious changes in the pattern of contractile proteins expressed (2). Hence, differentiation, as defined by contractility, appears to depend on the expression of protein(s), other than just the contractile proteins, whose expression is modulated by cell culture.
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Expression of PLC isoforms in VSM
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It is primarily the concentration of intracellular calcium that governs contraction and relaxation in VSM, and one mechanism by which calcium concentration can be increased is the mobilization of calcium from intracellular stores by IP3. In many cell types, PLC-ß isoforms are responsible for generation of IP3 from PIP2 after serpentine receptor activation. However, the expression of PLC-ß isoforms in VSM is a controversial subject. A number of groups have failed to detect this isoform, though others have described its presence in VSM. The discrepancy between these data may be due to species differences or even to intraspecies location differences within the vascular tree (Table 1).
Intriguingly, the profile of specific PLC isoforms expressed in VSM appears to be related to the state of differentiation. Although both PLC- and PLC- isoforms are routinely detected in VSM, a number of studies to date have indicated that PLC- is the predominant isoform of PLC in differentiated VSM. Indeed. Homma et al. (5) showed that, in cells freshly isolated from rabbit aorta, PLC-1 message and protein expression are clearly detectable. Although PLC-1 message levels remained consistent throughout the culture period, the level of protein expression appeared to decrease, which may be suggestive of a downregulation of PLC-1, corresponding with the cells dedifferentiating and taking on a synthetic phenotype. Indeed, this correlates nicely with work from Vincan et al. (13) that demonstrated a specific reduction in Gh protein expression in association with the phenotypic modulation of cultured VSM cells. Expression of PLC-2, on the other hand, was dramatically upregulated with culture, exhibiting a temporal response that also corresponds well with that of cell dedifferentiation (5).
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Discussion
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Considering the enzymatic activity of the PLC isoforms, it would be logical to think that the major function of the enzyme during intracellular signaling would result from the enzymatic hydrolysis of PIP2 to give IP3 and DAG. Clearly, the roles of PLC isoforms cannot be defined in such simple terms. PLC-ß isoforms are often absent from VSM, and a ubiquitous functional role is uncertain. The profile of PLC isoforms expressed in VSM also varies, depending on the state of differentiation. PLC-, the predominant isoform of PLC in differentiated contractile VSM, is associated with the actin cytoskeleton via RhoA and PIP2, and its activity is critically dependent on the presence of an intact PH domain. PLC- is associated with cell growth and proliferation; indeed, expression of this isoform is upregulated as VSM cells dedifferentiate and take on a synthetic phenotype. The mitogenic activity of this isoform may be as dependent on the presence of an intact SH domain and association with the actin cytoskeleton as it is on its catalytic activity.
The specificity of these isoforms for their proposed cellular functions was clearly demonstrated by disruption of the PLC-1 allele in mice, which results in a lethal mutation, with PLC-1 knockout embryos dying in midgestation (6). This demonstrates that the requirement for PLC-1 in the embryonic development of mice cannot be compensated for by any other tyrosine kinase-initiated signaling pathway or, more importantly in terms of this review, by any other isoform of PLC.
It is apparent that in VSM the differential regulation of specific PLC isoforms can be linked to the differential expression of these same isoforms. This, in its turn, may be related to the role of these isoforms in modulating specific cellular functions. Intriguingly, the role of PLC isoforms in the intracellular signaling pathways leading to either mitogenesis or contractility may be as much to do with their association with PIP2 and the actin cytoskeleton as with their catalytic function. Indeed, the hydrolysis of PIP2 by PLC isoforms may be just the tip of the iceberg in terms of the functions of a much-underrated family of enzymes.
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Acknowledgments
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We thank the British Heart Foundation for supporting the research carried out in our laboratory and Professor Sir David Weatherall for his faith and moral support through difficult times.
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References
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Introduction
Regulation of PLC isoforms
