The Sarcoplasmic Endoplasmic Reticulum Ca2ATPase Pump

SERCA is one of the modes that controls the homeostasis of intracellular calcium. SERCA2 transports Ca2+ into the lumen of the reticulum by an ATP-dependent mechanism. By virtue of this ability, SERCA2 plays an important part in normal physiological function such as the contraction and relaxation cycle of cardiac muscle, as well as in the pathogenesis of certain diseases. In addition, the correct localisation of the SERCA protein in the ER is of the utmost importance. As stated above, the localisation signal occurs in a stretch of 28 amino acids at the N-terminus of the molecule (Guerini et al. 1998). Ankyrin, a cytoskeletal protein, also seems to be involved in the targeting of SERCA. This is indicated by its abnormal localisation in ankyrin -/- mice. In these mutant mice, the absence of ankyrin also affects the localisation of ryanodine and inositol, 1, 4, 5-trisphophate (IP3) receptors (IP3R), both involved in calcium mobilisation (Tuvia et al. 1999). The ATP2A2 gene encodes the sarcoplasmic-endoplasm reticulum ATPase isoform type 2. Periasamy et al. (1999) have demonstrated the loss of calcium sequestering activity and consequent impairment of cardiac function in heterozygous ATP2A2 gene mutants.

Mutations of the ATP2A2 gene have been associated with the pathogenesis of Darier's disease (DD) (keratosis follicularis) (Sakuntabhai et al. 1999a, 1999b). DD is an autosomal dominantly inherited skin disorder. It is characterised by the presence of keratotic papules, and in histology is distinguished by acantholytic dyskeratosis. The keratotic papules occur mainly in the upper trunk, scalp, and palmar pits. Nail dystrophy is also a prominent feature of DD (Soroush and Gurevitch, 1997).

Because ATP2A2 controls calcium homeostasis in the cell, it is conceivable that its deregulation by mutation of the ATP2A2 gene might represent an early event in carcinogenesis. It was demonstrated many years ago that thapsigargin, an inhibitor of SERCA2, not only alters intracellular calcium levels, but also functions as a tumour promoter (Hakii, 1986; Thastrup et al. 1990). With the demonstration that ATP2A2 mutations are associated with the genesis of DD, there have also been several attempts to investigate the incidence of neoplasia in association with DD. Soroush and Gurevitch (1997) have pointed out that basal cell carcinomas and other skin neoplasms occur in patients with DD. Downs et al. (1997) described the occurrence of a subungual squamous cell carcinoma in a DD patient. However, in this case the human papilloma virus might have been the aetiological agent. In general, neoplasia may be associated only infrequently with DD.

There is an obvious common feature between DD and neoplasia that deserves a mention. This concerns the abnormal expression of the calcium-binding transmembrane glycoprotein cadherin. Some isoforms of cadherin have been regarded as invasion suppressors, because there is a significant body of evidence that associates the acquisition of invasive ability by tumour cells with the loss of cadherins. E-type cadherin is found in the cell membrane of epidermal cells. E-cadherin is distributed in the plasma membrane of keratinocytes, in the intercellular space between desmo-somes. Interestingly, this cadherin is expressed at greatly reduced levels in the acatholytic cells of DD and Hailey-Hailey disease (benign pemphigus) (Furukawa et al. 1997). Desmosomes provide the adhesion machinery in most epithelia, and abnormalities associated with them may be a key feature of inherited diseases such as DD and Hailey-Hailey disease. Indeed, the loss of cadherin may be the determining factor that causes the characteristic loss of adhesion between epidermal cells encountered in DD. Furukawa et al. (1997) also point out that both E- and P-cadherins are absent in squamous carcinomas, malignant melanomas and in Paget's disease. But basal cell carcinomas, which are noninvasive, do not show a loss of cadherin.

The above discussion underscores the importance of the SERCA2 calcium pump and intracellular calcium homeostasis in the calcium-signalling events associated with cell differentiation and dedifferentiation, and in the pathogenesis of DD and neoplasia.

Voltage-Gated Calcium Channels

The Voltage-gated calcium channels (VGCCs) are located in the plasma membrane. The high voltage-activated channel subtypes L,N, P, Q, and R occur as heterodimers of four subunits. The largest of the subunits is the al subunit, which spans the plasma membrane and the auxiliary subunits, p, y, and a2S (Isom et al. 1994). The properties of the calcium channel appear to be determined by the differential expression of al, which is the pore-forming subunit. The p subunit seems to regulate channel properties and the targeting of al. The p subunit, of which four isoforms (Pl-4) have been identified, is said to interact with al. This interaction is mediated by certain highly conserved domains (Pragnall et al. 1994; De Waard et al. 1994). The interaction between these subunits appears to regulate channel activity and the diversity of calcium currents (Varadi et al. 1991; Isom et al. 1994; Olcese et al. 1994). The p subunits are believed to modulate the kinetics of channel activation and inactivation by means of phosphorylation.

Calcium-binding proteins feature prominently in calcium homeostasis as important regulators of calcium channel activity. The elevation of intracellular levels of calcium can provide a negative feedback and close the channel. On the other hand, the feedback can have a positive element or facilitation, which opens the calcium channel. Zuhlke et al. (1999) have shown that CaM functions as a sensor for both negative and positive regulation of L-type channel activity. CaM appears to do this by binding to the consensus sequence called the IQ motif that occurs in the C-terminal a1C subunit of the channel protein. They substituted the isoleucine residue with alanine and reduced Ca2+-dependent inactivation and enhanced facilitation of the channel. Both inactivation and facilitation were abrogated when the isoleucine residue was converted to glutamate. CaM binds to the IQ motifs of other channel subtypes N, P/Q, and R as well (Peterson et al. 1999). Indeed, calcium-mediated modulation of P/Q channel subtypes also seems to involve CaM function. Again the channel activity is regulated CaM binding to the IQ motif of the a1A channel subunit (A. Lee et al. 1999).

The Deregulation of Calcium Homeostasis as a Primary Event in Carcinogenesis

The operation of ligand-gated calcium channels and the role that calcium binding proteins play in calcium homeostasis and as messengers of the calcium signal provide the major focus for this work. There is much justification for the view held by many investigators that the deregulation of calcium homeostasis in the cell might be a primary event in cell transformation and carcinogenesis. Indeed, several agents that block or retard the influx of calcium into cells appear to be able to inhibit the invasive ability of cancer cells, alter their adhesion properties, and inhibit tumour growth and stabilise the disease.

Carboxyamido-triazole (CAI) is one such compound. CAI has been reported to be able to inhibit the invasive behaviour of breast carcinoma cell lines in vitro. The matrix metalloproteinases associated with these cells also appear to be inhibited in parallel (Lambert et al. 1997). Not surprising, therefore, is the finding that CAI inhibits the adhesion of glioma cells to collagen type IV coated substrata and also inhibits their invasion in vitro. CAI also appears to be capable of inhibiting cell proliferation (Jacobs et al. 1997). The antiproliferation and anti-invasion properties of CAI have also been described by Wasilenko et al. (1996) using human prostate cancer cell lines. These are clear indications of the variety of downstream pathways activated by calcium influx that are inhibited by CAI. Finally, CAI has been reported to stabilise the disease in patients with a variety of refractory solid tumours (Kohn et al. 1996).

Verapamil, a selective L-type calcium channel blocker, also has demonstrable anti-proliferative and anti-invasive properties. These were recognised some years ago. Thus verapamil inhibits tumour growth in vitro (Brocchieri et al. 1996) and seems to arrest cells in the G0G1 phase of the cell cycle (Zeitler et al. 1997). The antiproliferative activity has been confirmed by Hoffman et al. (1998) on retinal pigment cells. Verapamil is able to inhibit the migration of CD4+ and CD8+ T lymphocytes across the endothelium (Blaheta et al. 2000). Other calcium antagonists such as mibefradil can also inhibit the adhesion and diapedesis of lymphocytes across the endothelium (Blaheta et al. 1998). Earlier Hailer et al. (1994) had noticed that the expression of endothelial adhesion molecules was unaltered by verapamil. It may be too simplistic to interpret these findings merely in terms of blocking of calcium channels. Nonetheless, some of these publications also indicate that the F-actin levels of the lymphocytes are reduced by the treatment. This could be due to changes in cytoskeletal dynamics that occur after verapamil-mediated modulation of intracellular calcium levels. This in turn could alter cellular motility. Other factors will have serious effects on the permeability of the endothelium. For instance, CD4+ lymphocytes form two subsets known as Th1 and Th2 cells. Upon activation both subsets produce a wide range of cytokines. Th1 cells secrete interleukin (IL)-2 and (IFN)-y, and Th2 cells secrete IL-4, 5, 10, and 13. Some of these lymphokines are bound to affect endothelial permeability. An additional factor that must be reckoned with is the generation of nitric oxide, which is known to inhibit the adhesion and spreading of endothelial cells. Nitric oxide also affects the formation of stress fibres in the cells, which is bound to influence cellular motility. Nitric oxide also actively influences angiogenesis. IFN-y is a powerful inducer of nitric oxide synthase (NOS), whereas IL-4 from Th2 cells inhibits NOS. It would be well to remember here that endothelial NOS is a Ca2+/CaM-inducible enzyme. It is activated when it is binds the Ca2+/CaM complex.

More recently, the effects of verapamil have been tested on the local invasion and metastasis of a murine carcinoma cell line in BALB/C mice. Farias et al. (1998) found that it inhibited local invasion and also produced a 50% reduction in both spontaneous and experimental metastasis. In common with CAI, the cytostatic and anti-invasive effects may be due to the inhibition of membrane-associated proteinases such as matrix metalloproteinases and urokinase-type plasminogen activator (Farias et al. 1998). These events are virtually terminal events in the process of inhibition of adhesion and invasiveness by verapamil. More upstream in the pathway, verapamil has been shown to down-regulate the expression of S100A4 (Parker and Sherbet, 1992), which, as discussed elsewhere in this book, is closely allied with p53 and stathmin genes in the control of cell cycle progression. Furthermore, S100A4 expression is closely linked with the remodelling of the extracellular matrix.

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