Antisense-mediated loss of calcium homoeostasis endoplasmic reticulum protein (CHERP; ERPROT213-21) impairs Ca2+ mobilization, nuclear factor of activated T-cells (NFAT) activation and cell proliferation in Jurkat T-lymphocytes.

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) P (3)-induced Ca(2+) release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca(2+) 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) P (3) receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lac I-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca(2+) (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 Ca(2+) concentration ([Ca(2+)](i)), but Ca(2+) influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5) P (3) receptors, but diminished the rise of [Ca(2+)](i) in response to PHA to </=30% of that in control cells, decreased Ca(2+) influx and slowed the initial rate of [Ca(2+)](i) rise caused by thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase, suggesting there was also some deficit in ER Ca(2+) stores. In CHERP-depleted cells the Ca(2+)-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 G(1) phase of the cell cycle. These findings provide further evidence that CHERP is an important component of the ER Ca(2+)-mobilizing system in cells, and its loss impairs Ca(2+)-dependent biochemical pathways and progression through the cell cycle.