Endoplasmic Reticulum Calcium Pumps and Tumor Cell Differentiation

Abstract

Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.

Keywords: SERCA; calcium signaling; cancer; differentiation; endoplasmic reticulum; ion transport; leukemia.

Figure 1

A simplified scheme of cellular calcium compartments, transporters and calcium ion fluxes. (A) Calcium mobilization during a cell activation event. Upon activation of plasma membrane receptors, phospholipase C (PLC)-induced hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) leads to the formation of the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Binding of IP3 on IP3-receptor calcium channels located in the ER membrane leads to channel opening and passive diffusion of calcium ions from the ER lumen into the cytosol (calcium release). Dissociation of calcium ions from the intraluminal part of STIM proteins located in the ER membrane leads to their interaction with Orai-type calcium channels located in the plasma membrane and Orai channel opening. Calcium influx through Orai channels (capacitative calcium influx) combined with calcium release from the ER leads to increased cytosolic calcium levels and the activation of calcium-dependent downstream signaling. (B) Elimination of cytosolic calcium ions after a cell activation event. Cytosolic calcium levels are decreased by the concerted action of SERCA, SPCA and PMCA-type calcium pumps, sodium/calcium exchangers (NCX) and the mitochondrial calcium uniporter complex (MCU). In non-muscle cells calcium is actively taken up in the ER by SERCA2b and SERCA3-type calcium pumps, and calcium (as well as manganese) transport into the Golgi complex is performed by SPCA-type pumps. Cytosolic calcium can also be taken up by mitochondria through the MCU and is eliminated from the cell through PMCA-type calcium pumps and sodium/calcium exchangers (NCX) into the extracellular medium that contains calcium ions in the low millimolar range. The concerted action of these mechanisms leads to the establishment of resting cytosolic calcium levels (low nanomolar). Calcium sequestered in the ER (in the high micromolar concentration range) can interact with STIM proteins, leading to inactivation of capacitative calcium influx, as well as with calcium-binding ER chaperones such as calreticulin.

Figure 2

Schematic representation of the evolution of SERCA2b and SERCA3 transport activities during an IP3-induced calcium release event from the endoplasmic reticulum. Calcium transport activity (the number of calcium ions transported by a pump molecule per unit time) of the pumps is stimulated by increasing calcium concentrations. However, due to its higher calcium affinity (K_Ca²⁺ ~ 0.2 µM), this stimulation occurs at lower calcium concentrations for SERCA2b than for the lower calcium affinity SERCA3 pump (K_Ca²⁺ ~ 1.2 µM). The calcium affinity of SERCA2b lies closer to the calcium concentration of a resting cell, whereas that of SERCA3 is closer to calcium levels that occur during activation, in particular in specialized ER microdomains, where calcium release is locally taking place. Consequently, whereas SERCA2b is nearly maximally active already in a resting cell, SERCA3-dependent transport is at that point only weakly active, becoming activated only later, during the peak of a calcium release event (when local cytosolic calcium concentrations near the ER approaches 1.2 µM). Note that at the beginning and at the end of the calcium signal (blue areas) neither SERCA2b nor SERCA3-dependent transport is stimulated significantly by calcium. When SERCA2b is replaced by SERCA3 during cell differentiation, calcium release is facilitated, allowing more robust calcium signals. The scenario is depicted in the absence of capacitative calcium influx (in the absence of extracellular calcium) for simplicity. Upward facing arrow: cytosolic calcium concentration, downward facing arrows: SERCA calcium transport intensity. Arrow lengths depict transport intensity relative to maximally stimulated (longest arrows); SERCA2b: grey, SERCA3: blue.

Figure 3

SERCA3 expression is lost in choroid plexus tumors. SERCA3 expression was investigated using immunohistochemistry with the 2H3 SERCA3-specific monoclonal antibody (Abnova) on deparaffinized, formalin-fixed sections of normal choroid plexus tissue (A), choroid plexus hyperplasia (B), grade I (C) and grade II papilloma (D) and choroid plexus carcinoma (E). Immunostaining was revealed using an avidin-biotin-peroxydase immunolabeling system with 3,3′diaminobenzidine as chromogen. Slides were counterstained with hematoxylin (blue). Whereas normal choroid plexus epithelial cells (A) and hyperplastic epithelium (B) express SERCA3 abundantly (brown coloration), expression in grade I and grade II papillomas (C,D) as well as in carcinoma (E) is barely detectable or absent. Original magnification: ×40.