We recently discovered a novel gene on chromosome 19p13.1 and its product, an integral endoplasmic reticulum (ER) membrane protein, termed CHERP (calcium homoeostasis endoplasmic reticulum protein). A monoclonal antibody against its C-terminal domain inhibits Ins(1,4,5)P3-induced Ca2+ release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca2+ mobilization by thrombin. In the present paper, we explore further CHERP's function in Jurkat T-lymphocytes. Confocal laser immunofluorescence microscopy showed that CHERP was co-localized with the Ins(1,4,5)P3 receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lacI-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca2+ (measured by fura-2 acetoxymethyl ester fluorescence) caused by phytohaemagglutinin (PHA) and thrombin. A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic free Ca2+ concentration ([Ca2+]i), but Ca2+ influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5)P3 receptors, but diminished the rise of [Ca2+]i in response to PHA to ≤30% of that in control cells, decreased Ca2+ influx and slowed the initial rate of [Ca2+]i rise caused by thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase, suggesting there was also some deficit in ER Ca2+ stores. In CHERP-depleted cells the Ca2+-dependent activation and translocation of the key transcription factor NFAT (nuclear factor of activated T-cells) from cytoplasm to nucleus was suppressed. Furthermore, cell proliferation was greatly slowed (as in HEL cells) along with a 60% decrease in cyclin D1, a key regulator of progression through the G1 phase of the cell cycle. These findings provide further evidence that CHERP is an important component of the ER Ca2+-mobilizing system in cells, and its loss impairs Ca2+-dependent biochemical pathways and progression through the cell cycle.
Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins
