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.Cellular calcium concentration gradients and calcium ion fluxes are important components of several signaling networks controlling cell growth, differentiation, or apoptosis (1, 2). In a resting cell, the cytosolic free calcium concentration is ϳ50 -100 nM, whereas the endoplasmic reticulum (ER) 1 or the extracellular medium contains calcium in the high micromolar to low millimolar range. Binding of several growth factors, hormones, chemokines, or bioactive peptides to their cell surface receptors leads to the formation of the second messenger inositol 1,4,5-trisphosphate (IP 3 ), which induces calcium release from the endoplasmic reticulum into the cytosol through IP 3 receptor calcium channels. The ensuing decrease of the calcium content of the ER lumen induces the opening of calcium channels in the plasma membrane, allowing calcium influx into the cytosol from the extracellular space. Calcium release from the ER and ensuing calcium influx lead to the augmentation of the cytosolic free calcium concentration. As m...

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/␤-catenin/T-cell factor 4 (TCF4) pathway and of specificity protein 1 (Sp1)-like factordependent 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 Calcium homeostasis of the endoplasmic reticulum (ER) is involved in several essential cell functions. Calcium stored in the ER is required for chaperone-assisted maturation of newly synthesized proteins transiting through the organelle. 1-3 Moreover, second messenger-induced calcium release from the ER through inositol-tris-phosphate-and ryanodine receptor-type calcium channels constitutes an integral part of many intracellular signal transduction pathways and networks. 4,5 Because ER calcium homeostasis is therefore involved in many constitutive or inducible cell functions, calcium accumulation into this organelle, assured by sarco/endoplasmic reticulum calcium transport ATPase (SERCA)-type calcium pumps is essential for numerous cellular activities such as secretion, neuronal plasticity, stress responses, proliferation, differentiation, or various forms of cell death. 6 -12 Three SERCA genes are known that code by alternative splicing several isoforms, the expression of which is tissue dependent and developmentally regulated. 13

In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ib␣ (GPIb␣). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIb␣ N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIb␣, ␣IIb, and ␤3 were normally present. Proteins involved in Ca 2؉ homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca 2؉ ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP 3 -R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal megakaryocytes cultured from peripheral blood CD34 ؉ cells with TPO. Significantly, megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production. IntroductionVon Willebrand disease (VWD) is the most common inherited disorder of the platelet-vessel wall interaction and involves both quantitative and qualitative defects of von Willebrand factor (VWF), a crucial mediator of platelet function and carrier of the FVIII protein. In type 1 and type 3 VWD, deficiencies or absence of VWF protein are responsible for the bleeding syndrome, but in type 2 disease a functionally abnormal protein or the specific lack of large multimers account for the VWD phenotype. 1,2 In hemostasis, glycoprotein Ib␣ (GPIb␣) mediates platelet attachment to exposed subendothelium by binding through its N-terminus to the A1 domain of VWF exposed within the subendothelial matrix. 3,4 In healthy subjects, soluble VWF multimers in plasma fail to gain access to their binding site on GPIb␣, accessibility being controlled by a disulfide-linked double-loop region just below the leucine-rich repeats of GPIb␣. 5 In type 2B VWD, mutations giving rise to a selective number of amino acid substitutions in the A1 domain provide gain of function to the VWF multimers which then spontaneously bind to platelets in suspension through a direct interaction with GPIb␣. [6][7][8] This often results in the loss of the largest multimers from plasma, although these may be at least partially preserved in some cases. 9 Bleeding results from platelets having blocked GPIb function despite a heightened ristocetininduced platelet agglutination in platelet function testing, and perhaps through the relative hemostatic inefficiency of the remaining small multimers. The thrombocytopenia that accompanies this disorder in some, a...