Béla Papp

Smooth Muscle Cell Cycle and Proliferation RELATIONSHIP BETWEEN CALCIUM INFLUX AND SARCO-ENDOPLASMIC RETICULUM Ca2+ATPase REGULATION

The role of Ca2+ influx in the regulation of the sarco-endoplasmic reticulum Ca2+ATPases (SERCA) associated with intracellular Ca2+ pools was investigated during smooth muscle cell (SMC) proliferation induced by platelet-derived growth factor (PDGF). We first defined that the previously described up-regulation of the SERCA2a isoform found in vascular SMC after a 24-h stimulation with PDGF (Magnier, C., Papp, B., Corvazier, E., Bredoux, R., Wuytack, F., Eggermont, F., Maclouf, J., and Enouf, J. (1992) J. Biol. Chem. 267, 15808-15815) was precisely associated with SMC entry into S phase as it appeared linked with [3H]thymidine incorporation. This was further confirmed by testing the effect of transforming growth factor-β1, which inhibited both aortic SMC proliferation associated with G1 cell cycle arrest and PDGF-induced SERCA2a up-stimulation. Then, we tested the role of Ca2+ influx by using SR 33805, a new Ca2+ channel blocker, which was characterized with regard to the voltage Ca2+ channel blocker nifedipine and the capacitative entry Ca2+ blocker SKF 96365. SR 33805 was found to be the most potent inhibitor of both PDGF-induced SMC proliferation and the associated rise in intracellular Ca2+ concentration with IC50 values of 0.2 ± 0.1 and 0.31 ± 0. 04 µM, respectively. Finally, by examining in parallel both SERCA2a and SERCA2b isoforms, in terms of activity and expression, we could determine that PDGF-induced stimulation of total SERCA activity (detected by formation of the phosphorylated intermediate, E∼P) and of SERCA2a expression (Western blotting) were abolished when extracellular Ca2+ entry was prevented by SR 33805. This study demonstrates that SERCA2a up-regulation is: 1) related to the G1/S transition step of cell cycle and 2) dependent on Ca2+ entry during PDGF-induced SMC proliferation.

The Loss of Sarco/Endoplasmic Reticulum Calcium Transport ATPase 3 Expression Is an Early Event during the Multistep Process of Colon Carcinogenesis

Calcium accumulation in the endoplasmic reticulum is accomplished by sarco/endoplasmic reticulum calcium transport ATPases (SERCA enzymes). To better characterize the role of SERCA3 in colon carcinogenesis, its expression has been investigated in colonic epithelium, benign lesions, adenomas, and adenocarcinomas. In addition, the regulation of SERCA3 expression was analyzed in the context of the adenomatous polyposis coli/beta-catenin/T-cell factor 4 (TCF4) pathway and of specificity protein 1 (Sp1)-like factor-dependent transcription. We report that SERCA3 expression increased along the crypts as cells differentiated in normal colonic mucosa and in hyperplastic polyps, was moderately and heterogeneously expressed in colonic adenomas with expression levels inversely correlated with the degree of dysplasia, was barely detectable in well and moderately differentiated adenocarcinomas, and was absent in poorly differentiated tumors. Inhibition of Sp1-like factor-dependent transcription blocked SERCA3 expression during cell differentiation, and SERCA3 expression was induced by the expression of dominant-negative TCF4 in colon cancer cells. These data link SERCA3 expression to the state of differentiation of colonic epithelial cells, and relate SERCA3 expression, already decreased in adenomas, to enhanced adenomatous polyposis coli/beta-catenin/TCF4-dependent signaling and deficient Sp1-like factor-dependent transcription. In conclusion, intracellular calcium homeostasis becomes progressively anomalous during colon carcinogenesis as reflected by deficient SERCA3 expression.

Characterization of the inositol trisphosphate-sensitive and insensitive calcium stores by selective inhibition of the endoplasmic reticulum-type calcium pump isoforms in isolated platelet membrane vesicles

In mixed platelet membrane vesicles the presence of two distinct endoplasmic reticulum-type calcium pump enzymes of 100 and 97 kD molecular mass has been demonstrated. We have previously shown that both calcium pumps were recognized by polyclonal anti-sarcoplasmic reticulum calcium pump antisera [11]. In the present work we studied the effects of several calcium pump inhibitors on active calcium transport and inositol trisphosphate-induced calcium release in these vesicles in an attempt to assign the two calcium pump isoenzymes to specific calcium pools. The effect of the PL/IM 430 inhibitory anti-calcium pump antibody was compared to that of other calcium pump inhibitors acting predominantly on the 100 and the 97 kD calcium pump isoforms, respectively. The PL/IM 430 antibody, which recognized the 97 kD pump on Western blots and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, which inhibited phosphoenzyme formation of the same pump isoform, inhibited calcium accumulation predominantly into an inositol trisphosphate-releasable calcium pool. On the other hand, low concentration of thapsigargin, which inhibited phosphoenzyme formation mainly of the 100 kD pump isozyme, had a more pronounced effect on calcium uptake into an inositol trisphosphate-resistant pool. These data suggest that in platelets the 97 kD calcium pump isoform is likely to be associated with the inositol trisphosphate-sensitive calcium storage organelle.

Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

The endoplasmic reticulum (ER) plays a key role in Ca(2+) signalling through Ca(2+) release via inositol 1,4,5-trisphosphate receptors (InsP(3)-Rs) and Ca(2+) uptake by sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL/IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a-c RNAs and InsP(3)-Rs using the in vitro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose-response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10(-8) M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-fold induction of SERCA3c, was observed after 24 h of PMA treatment. Isoform-specific Western blotting and/or reverse transcriptase PCR studies showed that InsP(3)-R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these InsP(3)-R types in PMA-induced MEG 01 cells revealed that: (i) InsP(3)-RI protein and mRNA showed no changes; (ii) InsP(3)-RII mRNA was up-regulated and peaked at hour 48 and (iii) InsP(3)-RIII mRNA and protein showed a transitory maximal 3- and 2.3-fold increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288-11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of InsP(3)-R types II and III was seen, but a distinct pattern of SERCA3 regulation was observed. These results suggest a profound reorganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca(2+)-dependent platelet functions.

Platelet Ca2+ATPases : A Plural, Species-Specific, and Multiple Hypertension-Regulated Expression System

Abstract —Gaining insight into nonmuscle Ca2+ signaling requires basic knowledge of the major structures involved. We investigated the expression of platelet Ca2+ATPases in normal and hypertension-associated abnormal Ca2+ signaling. First, overall identification of normotensive Wistar-Kyoto rat Ca2+ATPases was attempted by looking for newly described human platelet 3′-end alternatively spliced sarco/endoplasmic reticulum Ca2+ATPases (SERCA) 3b mRNA and plasma membrane Ca2+ATPase (PMCA) 1b and 4b proteins, in addition to SERCA2b and SERCA3a isoforms. For SERCAs, comparative analyses of human and Wistar-Kyoto rat SERCA3 platelet mRNA by reverse transcription–polymerase chain reaction (RT-PCR) followed by sequencing established that human platelets coexpressed SERCA3b and a third SERCA3c, while rat cells were devoid of them but expressed a still unknown splice variant that we termed rSERCA3b/3c. Its identification using 3′-end SERCA3 gene and rapid amplification of cDNA ends (RACE)–PCR studies showed that it results from an additional SERCA3 alternative splicing process, which uses a second alternative polyadenylation site located in the last intron. For PMCAs, with the use of gene-specific RT-PCR followed by sequencing and Western blotting using 5F10 monoclonal antibody, expression of human and rat platelet PMCA1b and PMCA4b was similar. Second, comparative analysis of these newly identified Ca2+ATPases and SERCA3a in age-matched spontaneously hypertensive rat platelets demonstrated (1) a marked downregulation of rSERCA3b/3c, which became null, and a 1.71-fold increase in SERCA3a and (2) an opposite regulation of the 2 PMCAs, namely, a 3.3-fold decrease in PMCA1b mRNA and a 3.7-fold increase in PMCA4b mRNA. Hence, platelets coexpress multiple, diverse, and species-specific Ca2+ATPases, including a novel fourth SERCA3. Moreover, expression of PMCA (1b and 4b), SERCA3a, and rSERCA3b/3c was modulated in rat hypertension. Hence, Ca2+ATPases should be regarded as constituting a new rational basis for the understanding of nonmuscle cell Ca2+ signaling.

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets

We recently identified a multi‐SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti‐Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti‐SERCA 2b, anti‐SERCA 3, anti‐SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α‐granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes.  Thus, the multi‐SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub‐compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

Expression of hPMCA4b, the major form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in mature human platelets.

Antibodies 5F10 and JA3 (raised against the erythrocyte Ca2+ pump) were used to identify hPMCA4b as the major form of the plasma membrane Ca2+ pump in human platelets and in three human megakaryoblastoid cell lines, MEG 01, DAMI and CHRF 288-11. 5F10 was used because it has been shown to recognize all known isoforms of the hPMCA and JA3 because it reacts exclusively with hPMCA4b [Caride A.J., Filoteo A.G., Enyedi A., Verma A.K., Penniston J.T. Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies. Biochem J 1996; 316: 353-359]. In addition to hPMCA4b, hPMCA1b was also detected in the megakaryoblastoid cells by using isoform-specific polyclonal antibodies. The apparent size of this isoform, however, was smaller than that seen in HeLa and COS-7 cell membranes indicating the presence of a modified form of hPMCA1b. In platelets, no evidence of the expression of hPMCA1b could be found. The amount of PMCA in these cells was compared with that of the constitutive form of the sarco/endoplasmic reticulum Ca2+ pump in non-muscle cells (SERCA2b) and also with the amount of PMCA in human erythrocytes. A very low level of the plasma membrane Ca2+ pump was found in platelets while in their precursor cells the expression of this Ca2+ pump was much more abundant. Whereas the expression level of PMCA decreased dramatically in mature human platelets, the expression of SERCA2b did not change substantially upon megakaryocytic differentiation.

Modulation of B-cell endoplasmic reticulum calcium homeostasis by Epstein-Barr virus latent membrane protein-1.

BackgroundCalcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated.ResultsTwo Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitts lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles.ConclusionThe data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.

Expression of two isoforms of the third sarco/endoplasmic reticulum Ca2+ ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody.

Human platelets express several sarco/endoplasmic reticulum Ca2+ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3′‐end part of platelet SERCA3 mRNA, by means of RT‐PCR amplification using sets of primers covering the N−3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N−1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down‐modulated in the platelet precursor megakaryocytic CHRF 288‐11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.

Multifaceted plasma membrane Ca2 + pumps: From structure to intracellular Ca2 + handling and cancer

Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.