Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676–684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166±26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Sarco/endoplasmic reticulum Ca²⁺-ATPases (SERCAs) pump Ca²⁺ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a–c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a–e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca²⁺ with similar affinities. However, they modulated the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and the endoplasmic reticulum Ca²⁺ content ([Ca²⁺]_er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca²⁺ signaling than previously appreciated.