PubMed · 12051037
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Mary Lord. 2002-05-27. Got calcium?. https://pubmed.ncbi.nlm.nih.gov/12051037/
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During liver regeneration, hepatocytes sense the damage and initiate proliferation of the quiescent cells through poorly understood mechanisms. Here, we have used cultured hepatic cells to study the roles played by intercellular calcium in mediating wound-healing processes. Well-differentiated and polarized Hep-G2 cells repaired an experimentally induced wound by induction of cell divisions. The resulting cellular growth did not occur evenly across the healing cell lawn; instead, proliferations were three times more active within 150-200 microm from the wound edge than further away; this periwound preferential cell growth was not observed in the poorly differentiated and/or nonpolarized cells. We have provided experimental evidence demonstrating that the wounding procedure itself could elicit a propagating calcium wave, and interestingly, blocking this injury-associated intercellular calcium communication could effectively inhibit the biased cell growth along the margin of the wound. A photolithography-based patterned cell culture system was employed to help delineate the mechanisms underlying this type of calcium signaling. In conclusion, our results suggested that intercellular communications via propagating calcium waves coordinate regenerative cell proliferations in response to hepatic tissue losses.
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.
Glutamate decarboxylase (GAD, EC 4.1.1.15) catalyses the alpha-decarboxylation of glutamate to produce gamma-aminobutyrate (GABA). The nucleotide sequences of two divergent GADs (designated GAD1 and GAD3) were isolated from a Nicotiana tabacum L. cv. Samsun NN leaf cDNA library. Open reading frames indicated that GAD1 encodes a polypeptide of 496 amino acids and has greater than 99% identity with known tobacco GADs, whereas GAD3 encodes a polypeptide of 491 amino acids and has about 14% divergence from known tobacco GADs. Genomic DNA analysis suggested that there are at least four tobacco GAD genes, existing in pairs of highly identical genes. An in vitro assay at pH 7.3 revealed that activities of the recombinant proteins, after isolation from Escherichia coli and partial purification by nickel-affinity chromatography, are 57-133 times the control levels in the presence of 0.5 mM calcium and 0.2 micro M bovine calmodulin.